Methods for rapid multiplexed amplification of target nucleic acids

ABSTRACT

A fast, multiplexed PCR system is described that can rapidly generate amplified nucleic acid products, for example, a full STR profile, from a target nucleic acid. Such systems include, for example, microfluidic biochips and a custom built thermal cycler, which are also described. The resulting STR profiles can satisfy forensic guidelines for signal strength, inter-loci peak height balance, heterozygous peak height ratio, incomplete non-template nucleotide addition, and stutter.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing date, under 35 U.S.C.§ 119(e), of U.S. Provisional Application Ser. No. 60/921,802 filed 4Apr. 2007; U.S. Provisional Application Ser. No. 60/964,502 filed 13Aug. 2007; and U.S. Provisional Application Ser. No. 61/028,073 filed 12Feb. 2008, each of that is hereby incorporated by reference in itsentirety. This application also incorporates by reference, in theirentireties, two U.S. Patent applications filed on even date herewith;the first entitled “INTEGRATED NUCLEIC ACID ANALYSIS”, Attorney DocketNo. 07-801-US; and the second entitled “PLASTIC MICROFLUIDIC SEPARATIONAND DETECTION PLATFORMS”, Attorney Docket No. 07-865-US.

FIELD OF THE INVENTION

The present invention generally relates to methods for the rapidamplification of one or more loci within a nucleic acid sample, as wellas thermal cyclers and systems useful for performing the methods.

BACKGROUND OF THE INVENTION

The polymerase chain reaction (PCR) is an enzymatic reaction thatfacilitates rapid exponential amplification of nucleic acid sequences invitro. In forensics, PCR can be utilized to identify individuals basedon the amplification of small regions of the human genome containing aclass of repeated DNA known as Short Tandem Repeats (STRs). The unitlength of a given STR repeat ranges between 2-10 base pairs, and STRsgenerally fall within non-coding and flanking sequences but occasionallywithin coding regions (Edwards et al., Am. J. Hum. Genet. 1991, 49,746-756). There are several hundred thousand STR loci in the humangenome, occurring on average every 6-10 kb (Beckman and Weber, Genomics1992, 12, 627-631) and appearing to be highly polymorphic (Edwards etal., Trans. Assoc. Am. Physicians 1989, 102, 185-194). STR analysis hasbecome a major tool in the forensic armamentarium with a growing set ofapplications including paternity testing, human identification in massdisasters, and routine typing of children.

While several commercially available STR kits have been developed forsynthesizing the desired PCR products with high specificity, there aresignificant areas in which current STR technologies can be improved.Most importantly, the average time to complete multiplex PCR usingcommercial STR typing kits is approximately 2.14 hours; thetime-consuming and labor-intensive nature of these assays hascontributed to backlogs in forensic laboratories. While the advent ofautomated instrumentation to simultaneously process multiple samples hashelped to alleviate a significant bottleneck in typing throughput, theincreasing number of samples to be analyzed will require furtheracceleration of the process. Furthermore, there is a need to increasethe sensitivity of STR assays as well as to improve the detection of theamplified products (Gill, Croat. Med. J. 2001, 42, 229-32). Currentlyavailable STR kits contain nine to sixteen loci and work is underway inthe field to increase the number of loci that can be detected. Certainapplications of STR analysis in the field can be conducted using 4 ormore loci.

PCR can also be applied in a wide range of clinical settings. Forexample, PCR can be utilized to diagnose bacterial infections such asthose caused by Group A Streptococci, methicillin resistant S. aureus,and vancomycin resistant Enterococci and is generally more sensitivethan culture-based diagnostic techniques. Fungal infections can besimilarly diagnosed. PCR can be used to diagnose respiratory viruses(e.g., respiratory syncytial virus, adenovirus, and influenza andparainfluenza viruses), _(B)enito-urinary viruses (e.g., herpes simplexvirus and typing human papilloma viruses), meningitis (e.g., herpessimplex virus, Epstein-Barr virus, varicella-zoster virus, andenteroviruses), and hepatitis (e.g., hepatitis B and C). PCR is alsouseful in preimplantation genetic diagnosis including the assessment ofaneuploidy as well as the diagnosis of inherited diseases. From oncologyto rheumatology and from hematology to gastroenterology, it would bedifficult to find an area of medicine not impacted by PCR.

PCR has also been applied in a variety of non-clinical settingsincluding veterinary identification (analogous to human STR typing),veterinary diagnostics, assessment of food safety, detection ofagricultural pathogens and pharmacogenomics. An application of growingimportance concerns the identification of biological weapons agents inclinical and environmental samples. Real-time PCR, a close relative ofPCR that allows quantitation of the amount of product present in areaction following each amplification cycle, is utilized in essentiallythe same applications as PCR itself (see, Espy et al., ClinicalMicrobiology Reviews 2006, 19, 1656-256).

Most commercially available thermal cycling instruments are limited inthat they receive temperature feedback directly from and control theblock temperature as opposed to the PCR solution temperature. As aconsequence, the temperature profile of the solution, which is criticalto the success of the PCR, is likely to be grossly different from thedesired profile. Moreover, much of the literature on increasing PCRspeed and sensitivity has focused on amplification of one particularlocus at a time (“singleplex assays”) and only limited success has beenreported in simultaneous amplification of multiple loci (“multiplexassays”) as required for forensic STR typing, clinical diagnostic andnon-clinical applications. For example, a 160 nL chamber coupled to anintegrated heater has been shown to be capable of amplification andseparation of 4 STRs contained in a Y-STR assay in 80 minutes with adetection limit of 20 copies of template DNA. (Liu et al., Anal. Chem.2007, 79, 1881-1889). Increased PCR sensitivity due to reduced PCRreaction volume has also been reported for the PowerlPlex® 16 System,although no attempt was made to increase reaction speed (Schmidt et al.,Int. J. Legal Med. 2006, 120, 42-48). However, neither report providedfor the significantly shorter amplification times needed in the art.Hopwood et al., (International Congress Series 1288 (2006) 639-641)report a one hundred minute amplification using a set of 11 STR primers.With respect to clinical diagnostics, a panel of seven commonrespiratory viruses was amplified using a nanochip system in a PCR assayrequiring 97.5 minutes (Takahashi et al., J. Clin. Microbiol 2008,doi:10.1128/JCM.01947-07).

Many of the applications of PCR (and real time PCR) such as forensichuman . identification by STR typing, clinical diagnostics, andbiological weapons agent detection are extremely time sensitive and manyof the applications are best performed in a multiplex setting. Inaddition, many of these applications are utilized in settings in whichlimited sample is available (e.g., a small number of pathogens from aclinical or environmental sample or a small number of human cells from aforensic sample) and sensitivity of the reaction is critical.

Notably, Horsman et al., (J. Forensic Sci., 2007, 52, 784-799) Id. at792 stated that “PCR has been a common pursuit among analyticalmicrochip researchers, as demonstrated by the wealth of literature onthe topic. However, for forensic DNA analysis, there remain a number ofavenues for development. Extensive work has not been shown using thecommercially available forensic STR kits or, further, multiple STRamplifications on a single device. When fully developed, however,microchip PCR will undoubtedly be a considerable time and cost savingsto the forensic community.” Therefore, there exists a need in the artfor rapid and sensitive methods to successfully provide simultaneousamplification of a plurality of loci within a nucleic acid sample for abroad range of applications.

SUMMARY OF THE INVENTION

The instruments, biochips, methods and systems of the invention providethe capability of heating and cooling a PCR solution rapidly,controllably, and reproducibly through monitoring and controlling thethermal cycler based on, at least in part, the actual temperature of thesolution. The inventive instruments, biochips, methods and systemsdisclosed herein provide the ability to monitor and/or accuratelycontrol the reaction temperature of a solution within a biochip to avoidover- or under-heating through the specific incorporation ofthermosensors that are not present in commercial thermal cyclers. Theability to rapidly heat and cool reaction solutions to such temperaturesallows ramping and settling times to be minimized and incubation time atthe desired temperature to dominate the total step time. Further, theinstruments, biochips, methods and systems of the invention providedherein impart the ability to rapidly alter and equilibrate thetemperature of a reaction solution, thereby greatly increasing the speedat which an amplification reaction may proceed.

Fast multiplex PCR amplification times as short as seventeen minuteshave been achieved using instruments, biochips, methods and systems ofthe invention. Additional time reductions are possible based on theteachings of this invention. Further, the fast PCR methods of theinvention are effective over a wide dynamic range, are extremelysensitive and are compatible with a wide range of commercially availableenzymes and reagents. For forensic applications, the instruments,biochips, methods and systems of the invention enable significantreductions in the time required to generate full profiles that satisfyinterpretation guidelines for STR analysis.

In a first aspect, the invention provides thermal cyclers comprising atemperature control element (TCE) wherein a first surface of said TCE isadapted to receive a sample chamber containing a solution and a sensingchamber containing a thermosensor, wherein the thermosensor providesfeedback to the TCE to set or maintain the sample at a desiredtemperature. In a second aspect, the invention provides thermal cyclersfurther comprising a second thermosensor positioned to monitor thetemperature of the first surface of the TCE.

In a second aspect, the invention provides systems comprising a biochipcomprising one or a plurality of reaction chambers comprising a portionof the biochip having a volume, wherein each reaction chamber furthercomprises a microfluidic inlet channel and a microfluidic outletchannel, wherein each reaction chamber is less than 200 μm from acontact surface of the biochip substrate; the system further comprisinga theimal cycler, comprising a temperature control element (TCE) whereina first surface of the TCE is adapted to receive a substrate containinga sample, and a thermosensor positioned to measure the temperature ofsample in the substrate and provide feedback to the TCE to set ormaintain the sample at a desired temperature said thermal cycler inthermal communication with the contact surface of the biochip substrate.In a third aspect, the invention provides systems comprising a biochip,comprising one or a plurality of reaction chambers, wherein eachreaction chamber comprising a portion of the biochip having a volume,further comprising a microfluidic inlet channel and a microfluidicoutlet channel, wherein each reaction chamber is less than 100 μm from acontact surface of the biochip substrate; and a thermal cycler,comprising a temperature control element (TCE) wherein a first surfaceof the TCE is adapted to receive a substrate containing a sample, and athermosensor positioned to measure the temperature of sample in thesubstrate and provide feedback to the TCE to set or maintain the sampleat a desired temperature, said thermal cycler in thermal communicationwith the contact surface of the biochip substrate.

In a fourth aspect, the invention provides methods for simultaneouslyamplifying of a plurality of loci in a nucleic acid solution comprisingproviding one or a plurality of reaction chambers wherein each reactionchamber comprises (i) a nucleic acid solution comprising at least onecopy of at least one target nucleic acid to be amplified; (ii) one ormore buffers; (iii) one or more salts; (iv) a primer set correspondingto each of the plurality of loci to be amplified; (v) a nucleic acidpolymerase; and (vi) nucleotides, sequentially thermally cycling thetemperature of the nucleic acid solution in each reaction chamberbetween a denaturing state, an annealing state, and an extension statefor a predetermined number of cycles at heating and a cooling rates ofabout 4-150° C./sec, to yield a plurality of amplified loci in eachreaction chamber in about 97 minutes or less.

In a fifth aspect, the invention provides methods for simultaneouslyamplifying of a plurality of loci in a nucleic acid solution comprisingproviding one or a plurality of reaction chambers wherein each reactionchamber comprises (i) a nucleic acid solution comprising at least onecopy of at least one target nucleic acid to be amplified; (ii) one ormore buffers; (iii) one or more salts; (iv) a primer set correspondingto each of the plurality of loci to be amplified; (v) a nucleic acidpolymerase; and (vi) nucleotides, sequentially theiiiially cycling thetemperature of the nucleic acid solution in each reaction chamber for apredetermined number of cycles at heating and a cooling rates of about4-150° C./sec, to yield a plurality of amplified loci in each reactionchamber in about 97 minutes or less.

In a sixth aspect, the invention provides methods for simultaneouslyamplifying 5 or more loci in a nucleic acid solution comprisingproviding one or a plurality of reaction chambers wherein each reactionchamber comprises (i) a nucleic acid solution comprising at least onecopy of at least one target nucleic acid to be amplified; (ii) one ormore buffers; (iii) one or more salts; (iv) a primer set correspondingto the 5 or more loci to be amplified; (v) a nucleic acid polymerase;and (vi) nucleotides, sequentially thermally cycling the temperature ofthe nucleic acid solution in each reaction chamber between a denaturingstate, an annealing state, and an extension state for a predeterminednumber of cycles at heating and a cooling rates of about 4-150° C./sec,to yield 5 or more amplified loci in each reaction chamber.

In a seventh aspect, the invention provides integrated biochips systemscomprising a biochip comprising at least two reaction chambers inmicrofludic communication, wherein a first reaction chamber is inthermal communication with a thermal cycler, comprising: a temperaturecontrol element (TCE) wherein a first surface of the TCE is adapted toreceive a substrate containing a sample, and a thermosensor positionedto measure the temperature of sample in the substrate and providefeedback to the TCE to set or maintain the sample at a desiredtemperature wherein a contact surface of the biochip is in thermalcommunication with the first surface of the thermal cycler; and a secondreaction chamber in fluid connection with the first reaction chamber andadapted for nucleic acid extraction, nucleic acid purification, pre-PCRnucleic acid cleanup, post-PCR cleanup, pre-sequencing cleanup,sequencing, post-sequencing cleanup, nucleic acid separation, nucleicacid detection, reverse transcription, pre-reverse transcriptioncleanup, post-reverse transcription cleanup, nucleic acid ligation,nucleic acid hybridization, or quantification, wherein the firstreaction chamber is less than 200 μm from a contact surface of thebiochip.

Specific preferred embodiments of the present invention will becomeevident from the following more detailed description of certainpreferred embodiments and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a photograph of an embodiment of the thermal cycler of theinvention.

FIG. 1B is a photograph showing an embodiment of a 16-lane microfluidicbiochip for use with the thermal cycler shown in FIG. 1A.

FIG. 2A is a graph showing the temperature profiles of block andreaction solution for one thermal cycle of the standard STR cyclingprotocol described herein (total cycling time: 145.1 minutes).

FIG. 2B is a graph showing the temperature profiles of block andreaction solution for one thermal cycle of the fast cycling protocoldescribed herein (total cycling time: 19.56 minutes).

FIG. 3 is a graph showing temperature profiles of the heat pump and thereaction solution for one thea inial cycle for a thermal cycler of theinvention using fast cycling conditions (total cycling time: 17.3minutes).

FIG. 4A is a graph showing the STR profile generated in biochipreactions according to the invention using 0.5 ng template DNA.

FIG. 4B is a graph showing the STR profile generated in tube reactionsaccording to the invention using 0.5 ng template DNA.

FIG. 5A is a graph showing the effect of DNA template level on signalstrength in biochip reactions.

FIG. 5B is a graph showing the effect of DNA template level on signalstrength in tube reactions.

FIG. 6A is a graph showing the effect of DNA template level onheterozygous peak height ratio (PHR) in biochip reactions.

FIG. 6B is a graph showing the effect of DNA template level on PHR intube reactions.

FIG. 7A is a graph showing the effect of DNA template level onnon-template nucleotide addition (NTA) in biochip reactions.

FIG. 7B is a graph showing the effect of DNA template level on NTA intube reactions

FIG. 8A is a graph showing the effect of DNA template level on stutterin biochip reactions.

FIG. 8B is a graph showing the effect of DNA template level on stutterin tube reactions

FIG. 9A is a graph showing the profile for biochip (top) and tubereaction (bottom) generated with the COfiler™ primer set using ingtemplate DNA.

FIG. 9B is a graph showing the profile for biochip (top) and tubereaction (bottom) generated with the Identifiler™ primer set using ingtemplate DNA.

FIG. 10 is a graph showing the profile for an embodiment of a sequencingreaction, as described in Example 5.

DETAILED DESCRIPTION OF THE INVENTION

In order to achieve fast multiplexed nucleic acid amplification, such asPCR, the invention provides thermal cycling instrumentation, reactionvessels, and reaction conditions that can be used to amplify a pluralityof loci within a target nucleic acid sample. As is illustrated by theexamples provided herein, fast thermal cycling methods of the inventioncan be perfoiiiied in microfluidic biochips using the thermal cycler ofthe invention and the methods described herein.

The methods provided by the invention are capable of rapid multiplexamplification in applications in addition to those utilizing thebiochips and thermal cyclers described herein. For example, the use ofthin walled tubes in conventional thermal cyclers (for example blockbased thermal cyclers and the Roche LightCycler™) and the use ofamplification methods other than temperature cycled PCR (for exampleisothermal PCR or rolling circle amplification) are specificallycontemplated.

The methods, biochips, and thermal cyclers provided by the invention arecapable of amplifying a plurality of loci in under 100 minutes within agiven nucleic acid solution present at amounts of at least 0.006 ng ofhuman genomic DNA (the approximate amount of DNA in a single nucleatedhuman cell) containing the target nucleic acid locus or loci). In otherembodiments the amplification occurs in less than 90 min., less than 80min., less than 70 min., less than 60 min., less than 50 min., less than40 min., less than 30 min., less than 20 min., less that 17.7 min., lessthan 15 min., less than 10 min., or less than 5 min.

In other embodiments, a plurality of loci within a bacterial, viral,fungal, animal, or plant-derived genome can be amplified starting fromat least one copy of the target nucleic acid locus or loci. For example,a sample to be analyzed can comprise less than 1000 copies, less than400 copies, less than 200 copies, less than 100 copies, less than 50copies, less than 30 copies, less than 10 copies or at least 1 copy of atarget nucleic acid prior to the multiplexed amplification reaction. Inaddition, less than a single genome equivalent of DNA can be utilizedfor amplification if the target nucleic acid locus is present in morethan one copy in the genome. Generally, at least two loci, and up toapproximately 250 loci can be simultaneously amplified within eachtarget nucleic acid in a sample according to the methods describedherein. Further, at least two loci and up to approximately 250 loci canbe simultaneously amplified in a plurality of target nucleic acidsaccording to the methods described herein.

The target nucleic acids utilized herein can be any nucleic acid, forexample, human nucleic acids, bacterial nucleic acids, or viral nucleicacids. The target nucleic acid sample can be, for example, a nucleicacid sample from one or more cells, tissues, or bodily fluids such asblood, urine, semen, lymphatic fluid, cerebrospinal fluid, or amnioticfluid, or other biological samples, such as tissue culture cells, buccalswabs, mouthwashes, stool, tissues slices, biopsy aspiration, andarcheological samples such as bone or mummified tissue. Target nucleicacids can be, for example, DNA, RNA, or the DNA product of RNA subjectedto reverse transcription. Target samples can be derived from any sourceincluding, but not limited to, eukaryotes, plants, animals, vertebrates,fish, mammals, humans, non-humans, bacteria, microbes, viruses,biological sources, serum, plasma, blood, urine, semen, lymphatic fluid,cerebrospinal fluid, amniotic fluid, biopsies, needle aspirationbiopsies, cancers, tumors, tissues, cells, cell lysates, crude celllysates, tissue lysates, tissue culture cells, buccal swabs,mouthwashes, stool, mummified tissue, forensic sources, autopsies,archeological sources, infections, nosocomial infections, productionsources, drug preparations, biological molecule productions, proteinpreparations, lipid preparations, carbohydrate preparations, inanimateobjects, air, soil, sap, metal, fossils, excavated materials, and/orother terrestrial or extra-terrestrial materials and sources. The samplemay also contain mixtures of material from one source or differentsources. For example, nucleic acids of an infecting bacterium or viruscan be amplified along with human nucleic acids when nucleic acids fromsuch infected cells or tissues are amplified using the disclosedmethods. Types of useful target samples include eukaryotic samples,plant samples, animal samples, vertebrate samples, fish samples,mammalian samples, human samples, non-human samples, bacterial samples,microbial samples, viral samples, biological samples, serum samples,plasma samples, blood samples, urine samples, semen samples, lymphaticfluid samples, cerebrospinal fluid samples, amniotic fluid samples,biopsy samples, needle aspiration biopsy samples, cancer samples, tumorsamples, tissue samples, cell samples, cell lysate samples, crude celllysate samples, tissue lysate samples, tissue culture cell samples,buccal swab samples, mouthwash samples, stool samples, mummified tissuesamples, autopsy samples, archeological samples, infection samples,nosocomial infection samples, production samples, drug preparationsamples, biological molecule production samples, protein preparationsamples, lipid preparation samples, carbohydrate preparation samples,inanimate object samples, air samples, soil samples, sap samples, metalsamples, fossil samples, excavated material samples, and/or otherterrestrial or extra-terrestrial samples. Types of forensics samplesinclude blood, dried blood, bloodstains, buccal swabs, fingerprints,touch samples (e.g., epithelial cells left on the lip of a drinkingglass, the inner rim of a baseball cap, or cigarette butts), chewinggum, gastric contents, saliva, nail scrapings, soil, sexual assaultsamples, hair, bone, skin, and solid tissue. Types of environmentalsamples include unfiltered and filtered air and water, soil, swabsamples from surfaces, envelopes, and powders.

For example, the methods herein can provide amplified nucleic acidsamples whose analysis yields data suitable for forensic interpretation,and in particular, data that satisfies forensic interpretationguidelines. Such guidelines include signal strength, inter-loci peakheight balance, heterozygous peak height ratio (PHR), incompletenon-template nucleotide addition (NTA), and stutter (Scientific WorkingGroup on DNA Analysis Methods, Short Tandem Repeat (STR) InterpretationGuidelines. Forensic Science Communications, 2000, 2(3)).

The phrase “fluid communication” as used herein, refers to two chambers,or other components or regions containing a fluid, connected together sothat a fluid can flow between the two chambers, components, or regions.Therefore, two chambers that are in “fluid communication” can, forexample, be connected together by a microfluidic channel between the twochambers, such that a fluid can flow freely between the two chambers.Such microfluidic channels can optionally include one or more valvestherein which can be closed or occluded, in order to block and/orotherwise control fluid communication between the chambers.

The term “poly(methyl methacrylate) or “PMMA,” as used herein, means thesynthetic polymers of methyl methacrylate, including but not limited to,those sold under the tradenames Plexiglas™, Limacryl™, R-Cast™,Perspex™, Plazcryl™, Acrylex ™, ACrylite™, ACrylplast™, Altuglas™,Polycast™ and Lucite™, as well as those polymers described in U.S. Pat.Nos. 5,561,208, 5,462,995, and 5,334,424, each of which are herebyincorporated by reference.

The term “polycarbonate” as used herein means a polyester of carbonicacid and glycol or a divalent phenol. Examples of such glycols ordivalent phenols are p-xylyene glycol, 2,2-bis(4-oxyphenyl)propane,bis(4-oxyphenyl)niethane, 1,1-bis(4-oxyphenyl)ethane,1,1-bis(oxyphenyl)butane, 1,1-bis(oxyphenyl)cyclohexane,2,2-bis(oxyphenyl)butane, and mixtures thereof, including but notlimited to, those sold under the tradenames Calibre™, Makrolon™,Panlite™, Makroclear™, Cyrolon™, Lexan™ and Tuffak™.

As used herein the term “nucleic acid” is intended to encompass single-and double-stranded DNA and RNA, as well as any and all forms ofalternative nucleic acid containing modified bases, sugars, andbackbones. The term “nucleic acid” thus will be understood to include,but not be limited to, single- or double-stranded DNA or RNA (and formsthereof that can be partially single-stranded or partiallydouble-stranded), cDNA, aptamers, peptide nucleic acids (“PNA”), 2′-5′DNA (a synthetic material with a shortened backbone that has abase-spacing that matches the A conformation of DNA; 2′-5′ DNA will notnormally hybridize with DNA in the B form, but it will hybridize readilywith RNA), and locked nucleic acids (“LNA”). Nucleic acid analoguesinclude known analogues of natural nucleotides that have similar orimproved binding, hybridization of base-pairing properties. “Analogous”forms of purines and pyrimidines are well known in the art, and include,but are not limited to aziridinylcytosine, 4-acetylcytosine,5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethylaminomethyluracil, inosine, N⁶-isopentenyladenine,1-methyladenine, 1-methylpseudouracil, 1-methyl guanine,1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine,3-methylcytosine, 5-methylcytosine, N⁶-methyl adenine, 7-methylguanine,5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,beta-D-mannosylqueosine, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid methylester, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil,2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid, and2,6-diaminopurine. DNA backbone analogues provided by the inventioninclude phosphodiester, phosphorothioate, phosphorodithioate,methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate,3′-thioacetal, methylene(methylimino), 3′-N-carbamate, morpholinocarbamate, and peptide nucleic acids (PNAs), methylphosphonate linkagesor alternating methylphosphonate and phosphodiester linkages(Strauss-Soukup, 1997, Biochemistry 36:8692-8698), and benzylphosphonatelinkages, as discussed in U.S. Pat. No. 6,664,057; see alsoOLIGONUCLEOTIDES AND ANALOGUES, A PRACTICAL APPROACH, edited by F.Eckstein, IRL Press at Oxford University Press (1991); AntisenseStrategies, Annals of the New York Academy of Sciences, Volume 600, Eds.Baserga and Denhardt (NYAS 1992); Milligan, 1993, J. Med. Chem.36:1923-1937; Antisense Research and Applications (1993, CRC Press). Thenucleic acids herein can be extracted from cells or syntheticallyprepared according to any means known to those skilled in the art; forexample, the nucleic acids can be chemically synthesized or transcribedor reverse transcribed from cDNA or mRNA, among other sources.

The term “via” as used herein means a through-hole formed in a solidmaterial to allow fluidic connection between the top and bottom surfacesof the material.

The terms “locus” and “loci” (plural) as used herein mean one or morespecific positions on one or more nucleic acids (e.g., one or morechromosomes), as defined herein.

The terms “STR locus” and “STR loci” as used herein means a nucleotidesequence consisting of a repeating pattern of two or more nucleotides ata given locus of a target nucleic acid. The repeating pattern can rangein length from 2 to 10 base pairs (bp), and is typically in thenon-coding intron region.

According to one aspect of the invention, a thermal cycler is providedhaving the capability of heating and cooling a reaction solutionrapidly, controllably, and reproducibly. An example of an embodiment ofthe thermal cycler of the invention is shown in FIG. 1A. The ability torapidly heat and cool the reaction solution temperatures allows rampingand settling times to be minimized and incubation time at the desiredtemperature to dominate the total step time, enabling minimization ofmultiplex cycling times.

High heating and cooling rates can be achieved by utilizing atemperature control element (TCE), either alone or in fluidcommunication with a heat sink. A TCE comprises a means for heating andcooling, a thermosensor, a controller that receives signals from thethennosensor, and a power supply. In a preferred embodiment, a firstsurface of the TCE can be adapted to receive a sample chamber containinga solution and a sensing chamber containing an additional thermosensor.In this setting, the thermosensor is positioned within the sensingchamber mounted to the TCE such that it simulates the conditions withinthe sample chamber. This sensing chamber is fabricated such that it hasthe same material stack-up as the sample chamber. A thermocouple mountedwithin the temperature sensor is embedded in the structure at ananalogous position to that of the sample in the sample chamber. Thissensor reports the effective temperature of the solutions in the samplechamber. Commercially-available Type-T or Type-K thermocouples (fromOmega Engineering, Stamford, Conn.) are most applicable but other typesof thermocouple and thermosensor may be used including thermisters,semiconductors, and infrared. The thermosensor within the sensingchamber provides feedback to the TCE to set or maintain the sample atthe desired temperature. In this way, the sample temperature can bemeasured indirectly and controlled without inserting a thermosensor intothe reaction chamber itself. Alternatively, a thermosensor can be placeddirectly into the reaction chamber and used to set and maintain sampletemperature, eliminating the need for the sensing chamber. As oneskilled in the art will appreciate, other types of sensors such aspressure sensors may be utilized according to the teachings of thisinvention.

The first surface of the TCE can be adapted to accept an essentiallyflat substrate by, for example, forming a recess in the first surfacefor accepting a substrate (e.g., a biochip, infra). Alternatively, theTCE can be adapted to accept one or more thin-walled tubes, defined astubes with wall diameters with regions less than 200 μm thick.Preferably, the heat sink is a high efficiency heat sink, such as, butnot limited to, fan-cooled heat sinks with copper bases and coolingfins. More preferably, the heat sink can be a fan cooled copper base andfins having a thermal resistance of about 0.4° C./W or less. Aparticular and non-limiting example of a high efficiency heat sink isE1U-N7BCC-03-GP (Coolermaster, Taiwan ROC).

The thermal cycler of the invention may further comprise a thermosensorpositioned to monitor the temperature of the first surface of the TCE.Additional thermosensors can be added as desired to achieve furtherimprovement in sample temperature control. The supplementarytemperatures that can be monitored include those on multiple regions onand within the substrate, multiple regions on and within the heat sink,cooling air input and output, sample input and output, and ambient.

Good thermal communication between the TCE and the heat sink is desired.When the two mating surfaces are properly prepared, intimate physicalcontact is sufficient to provide adequate thermal transfer between thetwo components. Thermal interface materials (TIMs) between the TCE andheat sink can be used to enhance thermal coupling. Such TIMs include butare not limited to adhesives, greases, phase-change materials (PCMs),metal thermal interface materials, ceramic thermal interface materials,soft metal alloys, indium, alumina nano-layer coatings, submicron films,glycol, water, oils, antifreeze, epoxy compounds, and others. Specificexamples include Arctic Silver or Ceramique (Arctic Silver, Visalia,Calif.; compounds that have thermal resistances of <0.007° C.-in²/W),compressible heat spring HSD4 (Indium Corp, Utica, N.Y.), HITHERM(GrafTech International Holdings Inc., Lakewood, Ohio), or directlybonding of the TCE to the surface of the heat sink. Thermal contact canbe further enhanced by physical clamping the components together with aaverage force of more than 2 psi, or more than 5 psi, or more than 10psi, or more than 30 psi, or more than 60 psi or more than 100 psi ormore than 200 psi, or by direct bonding of the surfaces.

Thermal transfer between the TCE and a substrate in contact therewithcan be increased with respect to block thermal cyclers, such as theEppendorf Mastercycler™ ep gradient S thermal cycler (which provide heatenergy via a silver block with high thermal conductivity and lowspecific heat capacity), by placing the substrate directly on the TCE.Suitable TCEs include, but are not limited to, a high heating andcooling capacity heat pump, and high power output Peltier devices;examples of Peltier devices are 9500/131/150B (Ferrotec, Bedford N.H.),XLT2393 (Marlow, Dallas Tex.). When utilized as a part of the TCE forthermal cyclers of the invention, Peltier devices are advantageouslypowered by an H-bridge. An example of an H-bridge device is the 5R7-001(Oven Industries).

When Peltier devices are used as a part of the TCE for thermal cyclersof the invention, it is advantageous to power the Peltier devices by anH-bridge with pulse width modulation for heating and cooling.Temperature feedback from the thermosensor which measures the sampletemperature drives the TCE to set and maintain the desired sampletemperature. Closed-loop temperature control algorithms for control ofthe TCE include, but are not limited to, PID control and fuzzy logiccontrol.

Said thermal controllers comprise a control algorithm that provides thecapacity for rapid transition from one target temperature state toanother target temperature state. This transition can be divided into 3distinct phases. In phase 1, there is a large difference between theactual temperature and target temperature (for example 1 to 20° C. orhigher). In this phase, ramping takes place at or near the maximum rateof the TCE device. In phase 2, the transition phase, the actualtemperature and target temperature are closer (less than approximately 1to 20° C.). In this case the controller must reduce the power to the TCEin order to prevent overshoot of the solution temperature and allow forrapid achievement of target temperature with minimal deviations andoscillations. In phase 3, the target temperature has been achieved andthe controller moderates power to the thermal cycler to maintain thesolution within a narrow range about the target temperature. Measurementof the temperature with the sensor provides more accurate feedback ofthe actual temperature and also allows the temperature of the TCEsurface temperature to be overdriven. Each of the above 3 phases may befurther subdivided into multiple sub-phases to provide for fasterresponse time, more accurate temperature control, increased stability,and increased tolerance to external variability.

In one example, the temperature of the substrate can be measured byplacing a thin thermocouple into a channel on the substrate surface. Inanother example, the second thermosensor can be housed in an enclosure,formed from essentially the same material as the substrate beingutilized, that holds the second thermosensor essentially the samedistance from the TCE as a reaction chamber on a substrate in contactwith the TCE. Such a second thermosensor can generally be separate fromthe substrate (i.e., a stand-alone sensor), and can be placed next tothe substrate on the first surface of the TCE.

The heat sink may, optionally, further comprise a variable speed coolingfan and/or a second heating element for controlling the temperature ofthe heat sink, where each additional element of the heat sink is incommunication with the second control element. This allows the coolingefficiency of the heat sink to be adjusted, in particular to keep theheat sink temperature essentially constant and independent ofenvironmental temperature changes. The heater can also precondition theheat sink to essentially the operating temperature.

To facilitate thermal coupling of a reaction solution in a substrate andthe TCE, uniform thermal communication of a contact surface of thesubstrate with the first surface of the TCE can be provided by applyinga force to the substrate to secure it thereto while the thermal cycleris in operation. Such forces are preferably applied by means that onlytemporarily hold the substrate to the first surface of the TCE and canbe readily removed upon completion of theimal cycling. For example, achip compression element (CCE) can be situated above the first surfaceof the TCE to allow the substrate to be placed between. The chipcompression element can then be engaged to hold the substrate in placeduring operation of the thermal cycler, and released to allow removal ofthe substrate. Proper integration of the CCE, TCE and heat sink allowsthe CCE to improve thermal coupling between and among all three of thesecomponents.

The portion of the CCE in contact with a substrate can be formed from alow thermal mass insulating material, including, but not limited to, afoam, for example WF71 Rohcell foam (Inspec foams, Magnolia, Ariz.). Forembodiments discussed herein the Rohacell is preferred. It has aspecific heat capacity of 1.4-1.6 (J/gK) [or less thermal mass] and athermal conductivity of 0.0345 W/mK (or less).

Biochip compression elements include, but are not limited to, one ormore clamps, springs, compressible foam, or a pressurized air bladderwhich can be inflated to provide force to hold the substrate onto thefirst surface of the TCE. Preferably, the chip compression elementprovides a substantially uniform force of about 5 to about 250 psia to asurface of the substrate, and more preferably, about 20 to about 50 psiato hold the substrate to the first surface of the TCE. Notably, thermalcommunication between the contact surface of the biochip substrate andthe TCE can be provided in the absence of a thermal coupling solutionssuch as thermal grease or glycol, although such can be utilized asnecessary.

The biochip compression elements provide a force on thebiochip-thermoelectric cooler-heat sink. This force serves to ensuregood thermal contact and hence heat transfer between the biochip and thetop surface of the TCE.

In one embodiment, the low thermal mass insulator is an air bladder andis utilized to provide the low thermal mass and insulating properties.In another embodiment, the low thermal mass insulator is a foam pad. Theclamping force can be applied to the foam pad by a pneumatic cylinder,or closed cell foam pads under compression or air pressure from an airbladder. In the latter case, the air bladder provides both theinsulation and the compressive force.

As described above, the thermal cycler can have a heating and/or coolingrate at the first surface of the TCE surface about 4-150° C. per second,and preferably about 8-150° C./sec, and more preferably about 10-150°C./sec. The thermal cycler can also have a heating or cooling rate at asolution within a reaction chamber of a substrate in uniform thermalcommunication with the first surface of the TCE (e.g., a biochip) ofabout 4-150° C. per second and preferably about 8-150° C./sec, and morepreferably about 10-150° C./sec. Further, the thermal cycler of theinvention can have a temperature stability of +/−1.0° C., and preferably+/−0.50° C., and more preferably +/−0.25° C.

Biochip

An embodiment of a biochip (i.e., a substrate for use with the thermalcycler of the invention) according to another aspect of the invention isshown for the sake of illustration in FIG. 1B as having 16 microfluidicsystems, each comprising an inlet and an outlet in fluid communicationwith each of the reaction chambers formed within the biochip. However,such disclosure is not intended to be limiting, rather, one skilled inthe art will readily recognize that the biochip can contain alternatenumbers of microfluidic systems (infra) including biochips with onesystem and biochips with two or more systems. The term “plurality” asused herein, means two or more, four or more, eight or more, 16 or more,32 or more, 48 or more, 64 or more, 96 or more, 128 or more, or 2-16,2-32, 2-48, 2-64, 2-96, 2-128, 8-128, 8 - 64, or 8-32 microfluidicchannels.

The biochip can comprise a substrate layer and a cover layer, where aportion of one or a plurality of microfluidic systems, comprisinggrooves and/or shaped depressions, are patterned into the substratelayer. A series of vias (i.e., through holes and/or inlets or outlets)can be formed in the cover layer to provide fluidic access to themicrofluidic channels and reaction chambers, and can be located at anylocation about the biochip. Alternatively, vias can be formed in thesubstrate layer instead of the cover layer to achieve the samefunctionality. The top surface of the substrate layer can be bonded withthe bottom surface of the cover layer to complete the microfluidicsystems. Techniques for fabricating polymer-based microfluidic systemsare reviewed extensively by Becker and Gartner (Becker, 2000,Electrophoresis 21, 12-26 and Becker, 2008, Electrophoresis 390, 89),which are hereby incorporated by reference in its entirety. Biochips canbe fabricated using materials such as unsaturated, partially unsaturatedor saturated cyclic olefin copolymers “COC”, unsaturated, partiallyunsaturated, or saturated cyclic olefin polymers “COP”, poly(methyl)methacrylate “PMMA”, polycarbonate “PC”, polypropylene “PP”,polyethylene “PE”, polyetheretherketone “PEEK”, poly(dimethylsiloxane)“PDMA”, polyimide “PI”. It is important to select a plastic with a glasstransition temperature greater than that of the maximal temperature tobe utilized in the amplification reaction. Any number of these processesand materials can be used to fabricate the biochips described herein. Inparticular, the biochips can be prepared by injection molding of aplastic substrate, for example, a COC or COP based polymers (currentlysold under the tradenames Topas™, Zeonex™, Zeonor™, and Apel™. In thisfabrication methodology, an injection mold and mold insert consisting ofthe negative of the features to be formed is fabricated by machining andsubsequent surface polishing. Together, the mold and insert allow thesubstrate layers to be fabricated and the formed substrate to comprisethe channels, reaction chamber features and vias. The substrate andcover layers can be diffusion bonded by the application of heat andpressure.

Alternatively, the biochips can be prepared by hot embossing of thinthermoplastic films with a master die of the negative of the structureto be produced. The master die can be prepared by using electroformingto replicate the device prepared in a solid substrate. The solidsubstrate can be glass sheets that are patterned by standardphotolithographic and chemical etching methods known to those skilled inthe art. The substrate and cover layers are diffusion bonded by theapplication of heat and pressure.

The substrate and cover layers of the biochip can be constructed from avariety of plastic substrates including, but not limited to,polyethylene, poly(acrylates) (e.g., poly(methyl methacrylate)),poly(carbonate)s, and unsaturated, partially unsaturated or saturatedcyclic olefin polymers (COP), or an unsaturated, partially unsaturated,or saturated cyclic olefin copolymers (COC). The thickness of plasticsubstrate and cover layers utilized in the present process is kept thinto minimize the mass thereof to thereby maximize thennal transferbetween the thermal cycler and the reaction solution contained in eachreaction chamber during their use. The plastic substrate and coverlayers can each, independently, have a thickness of less than 2 mm, lessthan 1 mm, less than 750 μm, less than 650 μm, less than 500 μm, lessthan 400 μm, less than 300 μm, less than 200 μm, or less than 100 μm; orplastic substrate and cover layers can each, independently, comprise aplastic having a thickness ranging from 25-2000 μm, 25-1000, 25-750 μm,25-650 mm, 25-500 μm, 25-400 μm, 25-300 μm, 25-200 μm, or 25-100 μm.Preferably, at least one of the substrate and cover layers has athickness of less than about 200 μm to maximize thermal transfer to thereaction solution contained in the reaction chambers of the biochip.More preferably, a contact surface of the biochip which is in contactwith the first surface of the TCE has a thickness of less than about 200μm.

Each reaction chamber can be foimed to have a volume of, for example,less than 100 μL. Preferably, each reaction chamber has a volume of lessthan about 50 μL, or less than about 40 μL, or less than about 30 μL, orless than about 25 μL, or less than about 20 μL, or less than about 15μL, or less than about 10 μL, or less than about 5 μL or less than about1 μL, or less than about 0.1 μL. Alternatively, each reaction chambercan be formed to have a volume ranging from about 0.1 μL to about 100 L.Preferably, each reaction chamber has a volume ranging from about 0.1 μLto about 10 μL or about 10 μL to about 50 μL. The reaction chambers aregenerally not coated with a polymer or silane coating. Reaction chambersmay be designed to have an inlet and an outlet channel. Alternatively, asingle channel may be used for inlet and outlet.

The biochip design of the invention leverages the benefits ofmicrofluidics including having a high surface to volume ratio andreduced diffusion times to maximize heat transfer, and uniforni heatingand cooling. The use of microfluidic technology also provides benefitswith respect to a fully-integrated forensic analysis instrument.Further, biochips fabricated by diffusion bonding, and without the useof adhesives to bond the various layers (e.g. COC layers), were testedand demonstrated to be capable of withstanding from 100 to 1500 psi ofpressure before failure based on the requirements of the desiredapplication. For example, the biochips of the present inventionwithstand 450 psi, sufficient for the desired thermal cyclingapplications.

It is noted that the specific embodiments of the biochips of theinvention set forth herein substantially lack any heating elementsintegrated into the biochip for heating and/or cooling the reactionchambers. Thermal cycling of the reaction chambers on the biochip isprovided externally, for example, by the thermal cycler of theinvention. Heating elements can be integrated into the biochips of thepresent invention, however.

In operation, one portion of the biochip can receive one or morereaction solutions, each independently comprising one or more reagents(e.g., for PCR) and or nucleic acid samples, through one or more inletsin fluid communication with one or more reaction chambers formed withinthe biochip. Simultaneous amplification of a plurality of samples can beperformed by injecting each of the nucleic acid samples in a separateseparation reaction chamber. An injector for simultaneously injecting aplurality samples into the plurality of sample or buffer wells can beprovided with the biochip to enable simultaneous multiple sampleamplification. Such injectors provide, for example, one sample of theplurality of samples to one reaction chamber of the plurality ofreaction chambers. Injectors can introduce the samples to the channelsaccording to any methods known to those skilled in the art, for example,by electrophoretic transport, pneumatic actuation or liquid actuationthrough a needle or tube or channel that connects the sample to thereaction chamber.

Following amplification (and optionally, nucleic acid extraction andquantification) the amplified nucleic acid product can be passed (e.g.,to a Genebench-FX™ 100) through one or more outlets in fluidcommunication with the reaction chambers for fragment separation andgeneration of STR profiles.

The relatively low cost of plastic manufacture allows the biochips ofthe invention to be disposable, eliminating the labor required to reusethe biochip and essentially eliminating the possibility ofcontamination. A single-use disposable would be particularlyadvantageous for low copy number analyses in that no possibility ofcontamination (other than initial sample collection) would exist. Insettings where neither contamination nor labor are major considerations,reusable plastic and glass biochips may be utilized.

Integration Methods

Using microfluidics allows fabrication of features to perform more thanone function on a single biochip. These functions can include nucleicacid extraction, nucleic acid purification, pre-PCR nucleic acidcleanup, post-PCR cleanup, pre-sequencing cleanup, sequencing,post-sequencing cleanup, nucleic acid separation, nucleic aciddetection, reverse transcription, pre-reverse transcription cleanup,post-reverse transcription cleanup, nucleic acid ligation, nucleic acidhybridization and quantification. Two or more of these functions can beconnected microfluidically to enable sequential processing of a sample;this coupling is termed integration.

One form of microfluidic DNA extraction can be achieved by inserting apurification medium between an input and output channel. Thispurification medium can be silica fiber based and use chaotropic-saltreagents to lyse the biological sample, expose the DNA and bind the DNAto the purification media. The lysate is then transported via the inputchannel through the purification medium to bind the DNA. Bound DNA iswashed by an ethanol based buffer to remove contaminants. This can beaccomplished by flowing wash reagents via the input channel through thepurification membrane. Bound DNA is then eluted from the membrane byflowing an appropriate low salt buffer (see, e.g., Boom, U.S. Pat. No.5,234,809) via the input channel through the purification membrane andout the output channel.

One approach to DNA quantification in a microfluidic foimat is basedupon real-time PCR. In this method of quantification, a reaction chamberis fabricated between an input and output channel. The reaction chamberis coupled to a thermal cycler and an optical excitation and detectionsystem is coupled to the reaction chamber to allow fluorescence from thereaction solution to be measured. The amount of DNA in the sample iscorrelated to the intensity of the fluorescence from the reactionchamber per cycle (see, e.g., Heid et al., Genome Research 1996, 6,986-994).

For further infoiivation about integration in microfluidic formats, seethe U.S. Patent application entitled “INTEGRATED NUCLEIC ACID ANALYSIS”filed on even day herewith (Attorney Docket No. 07-801-US), which ishereby incorporated by reference in its entirety. For furtherinformation about separation and detection in microfluidic formats seethe U.S. Patent application entitled “Plastic Microfluidic Separationand Detection Platfoinis” filed on even day herewith (Attorney DocketNo. 07-865-US which is hereby incorporated by reference in its entirety.

Microfluidic drives of the invention are means for transporting fluidswithin the reaction chambers of the integrated biochips. One type ofmicrofluidic drive is effected by incorporated a membrane pump whichtransports the fluid by sequential application of positive and negativepressure to the membrane. Alternatively, a positive displacement pumpcan be connected to the input of the microfluidic chamber. Adisplacement of the pump forces the fluid through the microfluidicchannel.

Integration can make use of microfluidic valves to gate fluid flowwithin the biochip. Valving can be accomplished with passive or activestructures. Passive valving structures include capillary valves thatstop fluid flow by utilizing capillary pressure. Fluids can flow throughthe capillary valving structure by the application of a pressure that issufficiently large enough to overcome the capillary forces. Activevalving structures include membrane valves which use flexible orsemi-rigid structures at a point between two channels. The applicationof pressure on the membrane causes it to close the channel. Theapplication of a vacuum to the membrane lifts it from the channel,allowing passage of fluids.

Amplification Methods

In yet another aspect, the invention provides methods for simultaneouslyamplifying a plurality of nucleic acid loci in one or more targetnucleic acids via rapid polymerase chain reaction (PCR). Such methodscomprise providing one or a plurality of reaction solutions to one or aplurality of reaction chambers, wherein each reaction solution comprises(i) at least one copy of at least one target nucleic acid, wherein eachtarget nucleic acid is the same or different and each target nucleicacid independently comprises a plurality loci to be amplified; (ii) oneor more buffers; (iii) one or more salts; (iv) a primer setcorresponding to the plurality of loci to be amplified; (v) a nucleicacid polymerase; and (vi) nucleotides. Each of the reaction solutions,for example, each of the target nucleic acids, can be the same ordifferent as necessary, for example, to run multiple simultaneousanalyses on the same nucleic acid sample, or to simultaneously runmultiple nucleic acid samples.

Each reaction chamber may be contained within a biochip of the inventionas described above or thin-walled reaction tubes. Thin-walled reactiontubes preferably have a wall thickness of less than about 200 μm.Preferably, thin-walled reaction tubes preferably have a wall thicknessof less than about 100 μm.

Primers for PCR amplification are oligonucleotide sequences that arespecifically designed to hybridize to loci of the target DNA. Theseprimers serve as starting points for polymerase extensions. Tofacilitate analysis of amplified fragments, labeled primers can also beused in PCR reactions. Labeled primers are oligonucleotide sequencesthat are coupled to a detectable moiety; a non-limiting example thereofis a fluorescent dye. When PCR is carried out with fluorescently labeledprimers, amplicons with a fluorescent label are generated. The methodsfor performing fast PCR are compatible with both labeled and unlabeledprimers, and fast multiplexed PCR have been demonstrated.

Primer sets can be any known to those skilled in the art for theamplification of a plurality of loci with a target nucleic acid, asdescribed above. For example, primers useful in amplification of one ormore loci in a human nucleic acid sample are described in U.S. Pat. No.5,582,989; U.S. Pat. No. 5,843,660; U.S. Pat. No. 6,221,598; U.S. Pat.No. 6,479,235; U.S. Pat. No. 6,531,282; and U.S. Pat. No. 7,008,771; andUS Patent Application Publication Nos. 2003/0180724; 2003/0186272; and2004/0137504, each of which are hereby incorporated by reference.

Further, primers useful in amplification of one or more loci in a viralnucleic acid sample are described in, for example, U.S. Pat. No.7,312,036; U.S. Pat. No. 6,958,210; U.S. Pat. No. 6,849,407; U.S. Pat.No. 6,790,952, and U.S. Pat. No. 6,472,155, each of which are herebyincorporated by reference.

Examples of primers useful in amplification of one or more loci in abacterial nucleic acid sample are described in U.S. Pat. No. 7,326,779;U.S. Pat. No. 7,205,111; U.S. Pat. No. 7,074,599; U.S. Pat. No.7,074,598; U.S. Pat. No. 6,664,080; and U.S. Pat. No. 5,994,066, each ofwhich are hereby incorporated by reference.

Salts and buffers include those familiar to those skilled in the art,including those comprising MgCl₂, and Tris-HCl and KCl, respectfully.Buffers may contain additives such as surfactants (e.g., Tweens),dimethyl sulfoxide (DMSO), glycerol, bovine serum albumin (BSA) andpolyethylene glycol (PEG), as well as others familiar to those skilledin the art. Nucleotides are generally deoxyribonucleoside triphosphates,such as deoxyadenosine triphosphate (dATP), deoxycytidine triphophate(dCTP), deoxyguanosine triphosphate (dGTP) and deoxythymidinetriphosphate (dTTP) are also added to the synthesis mixture in adequateamount for amplification of the target nucleic acid.

The solutions can be optionally heated to and held at a firsttemperature for a first period of time suitable for hot-start activationof the nucleic acid polymerases. Generally, the first period of time isless than about 90 seconds. The first temperature can be about 95 toabout 99° C. Polymerases with hot start mechanisms that can be activatedin 60 seconds or less include those utilizing antibody mediatedhot-start and aptmer mediated hot start mechanisms. Alternatively,hot-start polymerases need not be utilized in the present invention.

Subsequently, the temperature of the reaction solutions are sequentiallycycled between a denaturing state, an annealing state, and an extensionstate for a predetermined number of cycles. Generally, the one or aplurality of reaction solutions are cooled from the denaturing state tothe annealing state at a first cooling rate of about 1 to about 150°C./sec, or about 1 to about 100° C./sec; or about 1 to about 80° C./sec;or about 1 to about 60° C./sec; or about 1 to about 40° C./sec; or about1 to about 30° C./sec; or about 1 to about 20° C./sec; about 4 to about150° C./sec, or about 4 to about 100° C./sec; or about 4 to about 80°C./sec; or about 4 to about 60° C./sec; or about 4 to about 40° C./sec;or about 4 to about 30° C./sec; or about 4 to about 20° C./sec; or about10 to about 150° C./sec; or about 10 to about 100° C./sec; or about 10to about 80° C./sec; or about 10 to about 60° C./sec; of about 10 toabout 40° C./sec; or about 10 to about 30° C./sec; or about 10 to about20° C./sec. The one or a plurality of reaction solutions can be heatedfrom the annealing state to the extension state at a first heating rateof about 1 to about 150° C./sec, or about 1 to about 100° C./sec; orabout 1 to about 80° C./sec; or about 1 to about 60° C./sec; or about 1to about 40° C./sec; about 1 to about 30° C./sec; about 1 to about 20°C./sec; 4 to about 150° C./sec, or about 4 to about 100° C./sec; orabout 4 to about 80° C./sec; or about 4 to about 60° C./sec; or about 4to about 40° C./sec; about 4 to about 30° C./sec; about 4 to about 20°C./sec; or about 10 to about 150° C./sec; or about 10 to about 100°C./sec; or about 10 to about 80° C./sec; or about 10 to about 60°C./sec; of about 10 to about 40° C./sec; or about 10 to about 30°C./sec; or about 10 to about 20° C./sec; and/or the one or a pluralityof reaction solutions are heated from the extension state to thedenaturing state at a second heating rate of about 1 to about 150°C./sec, or about 1 to about 100° C./sec; or about 1 to about 80° C./sec;or about 1 to about 60° C./sec; or about 1 to about 40° C./sec; about 1to about 30° C./sec; about 1 to about 20° C./sec; about 4 to about 150°C./sec, or about 4 to about 100° C./sec; or about 4 to about 80° C./sec;or about 4 to about 60° C./sec; or about 4 to about 40° C./sec; about 4to about 30° C./sec; about 4 to about 20° C./sec; or about 10 to about150° C./sec; or about 10 to about 100° C./sec; or about 10 to about 80°C./sec; or about 10 to about 60° C./sec; of about 10 to about 40°C./sec; or about 10 to about 30° C./sec; or about 10 to about 20°C./sec. Finally, the reaction solutions are held at a final state toprovide one or a plurality of ampli tied nucleic acid products.

Denaturing states can range generally include from about 90 to 99° C.for times ranging from about 1 to 30 seconds. The actual times andtemperatures are enzyme, primer and target dependent. For the AppliedBiosystems (AB) multiplexed STR kit for amplifying human genomic DNA,about 95° C. for about 5 sec. being preferred.

The annealing temperature and time influence the specificity andefficiency of primer binding to a particular locus within a targetnucleic acid and are particularly important for multiplexed PCRreactions. The correct binding of a complete set of primer pairs duringthe annealing step can allow production of multiplexed amplification ofa plurality of loci, for example, one or a plurality of full STRprofiles with acceptable PHR and inter-locus signal strength balance.For a given primer pair, annealing states can range from about 50° C. to70° C. and times from about 1 to 30 seconds. The actual times andtemperatures are enzyme, primer, and target dependent. For the ABmultiplexed STR kit for amplifying human genomic DNA, about 59° C. for15 seconds is preferred.

Extension temperature and time primarily impact the allele product yieldand are an inherent property of the enzyme under study. It should benoted that the extension rates reported by the manufacturer are oftenprovided for singleplex reactions; extension rates for multiplexreactions can be much slower. For a given enzyme, extension states canrange from about 60 to 75° C. and times from about 1 to 30 seconds. Theactual times and temperatures are enzyme, primer, and target dependent.For the AB multiplexed STR kit for amplifying human genomic DNA, about72° C. for about 5 seconds is preferred. Preferably, for continuing apredetermined number of cycles, the reaction solution is heated from theextension state to the denaturing state at a third rate of about 1 toabout 150° C./sec, or about 1 to about 100° C./sec; or about 1 to about80° C./sec; or about 1 to about 60° Clsec; or about 1 to about 40°C./sec; or about 1 to about 30° C./sec; or about 1 to about 20° C./sec;4 to about 150° C./sec, or about 4 to about 100° C./sec; or about 4 toabout 80° C./sec; or about 4 to about 60° C./sec; or about 4 to about40° C./sec; or about 4 to about 30° C./sec; or about 4 to about 20°C./sec; or about 10 to about 150° C./sec; or about 10 to about 100°C./sec; or about 10 to about 80° C./sec; or about 10 to about 60°C./sec; of about 10 to about 40° Clsec; or about 10 to about 30° C./sec;or about 10 to about 20° C./sec. Generally, the predetermined number ofcycles is chosen to be about 10 to about 50 cycles, although fewer ormore cycles may be used as necessary.

Final extension times can be reduced significantly until incomplete NTAbegins to increase. For a given enzyme, final extension temperatures canrange from about 60 to 75° C. and times from about 0 to 300 seconds. Theactual times and temperatures are enzyme, primer, and target dependent.For the AB multiplexed STR kit for amplifying human genomic DNA, about72° C. for about 90 seconds is preferred.

In addition to the 3-step thermal cycling approach set forth above, thisprocess is also amenable to 2-step thermal cycling approaches. In thisapproach, the reaction solutions are sequentially cycled between adenaturing state, and an annealing/extension state for a predeterminednumber of cycles. This approach utilizes primers designed to anneal atthe extension temperature, allowing the annealing and extension steps toshare the same temperature. The reduced number of temperature transitionresults in a further reduction in the cycle time.

In certain embodiments, a plurality of amplified nucleic acid productscan be obtained in about 5 to about 20 minutes. In certain otherembodiments, a plurality of amplified nucleic acid products can beobtained in about 5 to 10 minutes, about 1 to 5 minutes, or less than 5minutes. Each amplified nucleic acid product can be generated startingfrom less than about 10 ng of a target nucleic acid. Preferably,amplified nucleic acid products can be generated starting from less thanabout 5 ng or less than about 2 ng of nucleic acid, or less than about 1ng of nucleic acid, or less than about 0.5 ng of nucleic acid, or lessthan about 0.2 ng of nucleic acid, or less than about 0.1 ng of nucleicacid, or less than about 0.05 ng of nucleic acid, or less than about0.006 ng of nucleic acid.

In other embodiments, such as the identification of biological weaponsagents in clinical or environmental samples or the diagnosis ofbacterial, viral, or fungal infections in humans, plants, and animals,amplified nucleic acid products can be generated starting from at leastone copy of a target nucleic acid. For example, a sample to be analyzedcan comprise less than 1000 copies (e.g., 1-1000 copies), less than 400copies, less than 200 copies, less than 100 copies, less than 50 copies,less than 30 copies, less than 10 copies or 1 copy of a target nucleicacid prior to the multiplexed amplification reaction.

In addition, less than a single genome equivalent of DNA can be utilizedfor amplification if the target nucleic acid locus is present in morethan one copy in the genome.

In any of the preceding methods, the theiinal cycling can be perfoimedfor a predetermined number of cycles to achieve sufficient amplificationof the loci in the target nucleic acid as can be readily deteiiuined byone skilled in the art. For example, the predetermined number of cyclescan range between about 10 and about 50 cycles, and preferably betweenabout 20 and 50 cycles. Further, in any of the preceding methods, atleast 2 loci of one or a plurality of nucleic acids can besimultaneously amplified. Depending on the desired application, greaterthan four, 5 to 10, 10 to 20, 20 to 30 or about 10 to 250 loci aresimultaneously amplified For example, for amplification of STR loci,10-20 loci may be preferred.

Preferably, the temperature of the reaction solutions is cycled by athermal cycler of the invention (supra). While it can be possible toutilize commercial block thermal cyclers for fast thermal cycling by thecompensating for lagging response of the PCR solution by setting theblock temperature higher than the desired solution temperature onheating steps and setting the block temperature lower than the desiredsolution temperature on cooling steps, this mode of operation iscumbersome to implement as the temperature setpoints required tocompensate for the slow ramping response must be determined empirically.Furtheiniore, as feedback and control are still performed by the blockand no monitoring of the solution temperature takes place, therepeatability and reproducibility of the profile can be influenced byexternal factors including the changes in the room temperature. Hence,the temperature profile of the solution is not reproducible.

Many commercially available polymerases can be adapted for use in fastPCR applications using the approaches described here. Typically, thenucleic acid polymerase has an extension rate of at least 100 bases/sec.A large number of polymerases available for PCR amplification includingTherms aquaticus (Taq), Pyrococcus furiosus (Pfu), Pyrococcus woesei(Pwo), Thermas flavus (Tfl), Themus thermophilus (Tth), Therms litoris(Tli) and Thermotoga maritime (Tma). These enzymes, modified version ofthese enzymes, and combination of enzymes, are commercially availablefrom vendors including Roche, Invitrogen, Qiagen, Strategene, andApplied Biosystems. Representative enzymes include PHUSION (New EnglandBiolabs, Ipswich , Mass.), Hot MasterTae™ (Eppendorf), PHUSION Mpx(Finnzymes), PyroStart (Fermentas), KOD (EMD Biosciences), Z-Taq(TAKARA), and CS3AC/LA (KlenTaq, University City , Mo.). A widely usedenzyme for PCR amplification for STR typing is the Taq polymerase, andthe TaqGold variant is supplied with the Identifiler™, Profiler™, andCOfiler™ kits.

In certain embodiments, the PCR conditions presented here can generatefull STR profiles from a human target nucleic acid with high efficiency,although production of a full profile is not required. A full profilefor autosomal STR can comprise loci such as amelogenin, D8S1179, D21S11,D7S820, CFSIPO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA,TPDX, D18S51, D5S818, FGA, or a plurality thereof. Other STR lociincluding mini-STRs, and Y-STR analysis. The criteria for optimizationof the protocols include the generation of full profiles, signalstrength, dynamic range, inter-locus signal strength balance, PHR,incomplete NTA, stutter, and total cycle time.

According one embodiment, protocols using the SpeedSTAR enzyme and thethermal cycler of the invention can reduce the total cycling time forbiochip and tube reactions to 17.3 and 19.1 min respectively, togenerate full STR profiles. In the protocol, the denaturing state isabout 98° C. for about 4 seconds, the annealing state is about 59° C.for about 15 seconds, the extension state is about 72° C. for about 7seconds, and the final state is about 70° C. for about 90 seconds.

In certain embodiments, the total cycling time for at least 10, 20, or30 multiplexed PCR cycles can range from about 1 minute to about 90minutes. Preferably, total cycling time for at least 10, 20, or 30multiplexed PCR cycles ranges from about 1 minute to about 90 minutes;or from about 1 minute to about 85 minutes; or from about 1 minute toabout 80 minutes; or from about 1 minute to about 75 minutes; or fromabout 1 minute to about 70 minutes; or from about 1 minute to about 65minutes; or from about 1 minute to about 60 minutes; or from about 1minute to about 55 minutes; or from about 1 minute to about 50 minutes;or from about 1 minute to about 45 minutes; or from about 1 minute toabout 40 minutes; or from about 1 minute to about 35 minutes; or fromabout 1 minute to about 30 minutes; or from about 1 minute to about 25minutes; or from about 1 minute to about 20 minutes; or from about 1minute to about 15 minutes; or from about 1 minute to about 10 minutesor from about 1 minute to about 5 minutes. In other embodiments, thetotal cycling time for at least 10, 20, or 30 multiplexed PCR cycles isless than about 90 minutes. Preferably, the total cycling time for atleast 10, 20, or 30 multiplexed PCR cycles is less than about 89, 85,80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, 4, 3, 2,or 1 minute.

The invention contemplates an integrated biochip comprising one or aplurality of microfluidic systems, for performing the multiplexed PCRamplification of a plurality of loci within a nucleic acid sample aswell as at least one other sample preparation and/or analysis methodwithin the same biochip platform. For example, within each microfluidicsystem on a biochip, each having a flow direction from an inlet port toan outlet port, the system can comprise a plurality of reactionchambers, wherein a first reaction chamber of the plurality of reactionchambers is in fluid communication with the inlet port, and an ultimatereaction chamber of the plurality of reaction chambers is in fluidcommunication with the outlet port, and at least one microchannelfluidly connecting each consecutive pair of reaction chambers along theflow direction. At least one reaction chamber in each microfluidicsystem can be less than 200 μm from a contact surface of the biochipsubstrate to facilitate thermal communication with a thermal cycler ofthe invention for performing multiplexed PCR within said reactionchamber.

Each of the remaining reaction chambers within each of the microfluidicsystems of the biochip can be adapted for nucleic acid extraction,nucleic acid purification, nucleic acid hybridization, nucleic acidligation, pre-PCR nucleic acid cleanup, post-PCR cleanup, pre-sequencingcleanup, sequencing, post-sequencing cleanup, separation and detection,reverse transcription, pre-reverse transcription cleanup, and/orpost-reverse transcription cleanup, electrophoretic separation, nucleicacid detection. The term “cleanup” as used herein means the removal ofreaction components (including anions, cations, oligonucleotides,nucleotides, preservatives, enzymes, or inhibitors) that may interferewith any of the reaction chamber processes listed above.

The Examples which follow are illustrative of specific embodiments ofthe invention, and various uses thereof. They set forth for explanatorypurposes only, and are not to be taken as limiting the invention.

EXAMPLES Example 1 Custom Thermal Cycler and Microfluidic Biochip

A thermal cycler of the invention, as shown in FIG. 1A, was used toperform fast cycling by allowing the PCR reaction solution temperaturesto be heated and cooled rapidly, controllably, and reproducibly. Thisinstrument accepts a 16-chamber microfluidic biochip and consists of ahigh output thermoelectric cooler/heater mounted to a high efficiencyheat sink. Each of 16 PCR reaction solutions was placed into anindividual chamber of the microfluidic biochip, coupled to the heat pumpby applying a 0.2 MPa of compressive pressure with a clamping mechanism.FIG. 1B shows a photograph of the 16-sample disposable plasticmicrofluidic biochip. Each PCR chamber is 500 μm deep and approximately1 mm wide and holds 7 μl of PCR reaction solution.

Instrumentation and Temperature Profiles

In the following examples, all amplification reactions in tubes wereperformed with an Eppendorf Mastercyler™ ep gradient S (Eppendorf NorthAmerica, Westbury, N.Y.). Block temperature profiles of the aboveinstrument were obtained using a 127 μm diameter type K thermocouplesensor which was attached directly to the block. For reaction solutionprofiles a 127 μm diameter type K thermocouple sensor was placed in the20 μL reaction solution, within a thin-walled PCR tube. Data acquisitionwas performed with an Omega HH506RA Multilogger thermometer set toacquire data at a rate of 100 Hz.

Amplification reactions in biochips were perfoimed using the thermalcycler of Example 1 with 16-sample plastic biochips as the reactionvessels. The solution temperature within the microfluidic biochip wasmonitored by inserting a thermocouple into a sensing chamber within thebiochip.

PCR —Reaction Mix Components and Cycling Conditions

Multiplex PCR reactions were performed with the AmpFCSTR® Profiler Plus®ID PCR Amplification Kit (Profiler Plus ID kit) (Applied Biosystems,Foster City, Calif.) using 9947A genomic DNA (Promega, Madison, Wis.) asa template. Polymerases used for amplification were either AmpliTaqGold® DNA Polymerase (TaqGold™) supplied with the Profiler Plus ID kitor other polymerases: SpeedSTAR HS DNA Polymerase (SpeedSTAR) (TakaraBIO USA Inc., Madison, Wis.), KOD Hot Start DNA Polymerase (KOD) (EMDBiosciences Inc., Gibbstown, N.J.), or PyroStartmi Fast PCR Master Mix(PyroStart) (Fermentas Inc., Glen Burnie, Md.). Multiplex PCRs withother polymerases were carried out using the labeled multiplex primerset from the Profiler Plus ID kit™ in combination with the polymerasespecific buffers and dNTPs. All tube PCRs were carried out in 0.2 mLthin-walled PCR tubes (Eppendorf North America, Westbury; NY) using theEppendorf Mastercyler™ ep gradient S. All biochip reactions wereamplified in the thermal cycler of FIG. 1A using 16-sample biochips.

The following PCR reaction mixtures were prepared and used for thermalcycling:

Standard TaqGold™ reactions:

Standard TaqGold™ multiplex reactions consisted of 9.55 μL Profiler PlusID reaction mix, 1 ng 9947A genomic DNA, 5 μL Profiler. Plus ID Primerset and 2.25 U TaqGold™ in a 25 μL reaction volume. Cycling conditions(block temperatures and times) were chosen following the manufacturersrecommendations and set to an initial 95° C. for 11 min (hot start)followed by 28 cycles of 1 min at 94° C. (denaturing), 1 min at 59° C.(annealing), 1 min at 72° C. (extension) and a final extension of 45 minat 60° C. Optimized TaqGold™ reactions:

TaqGold™ reactions optimized for fast cycling were carried out in a 10μL reaction volume containing 3.82 μL Profiler Plus ID reaction mix, 1ng 9947A genomic DNA, 2 μL Profiler Plus ID Primer set and 0.9 UTaqGold™. Reactions were cycled at 95° C. for 11 min, 28 cycles 10 s,98° C.; 45 s, 59° C.; 30 s, 72° C. and a final extension of 15 min at72° C.

SpeedSTAR Tube Reactions:

SpeedSTAR PCR mix components for tube PCR were: 2 μL Profiler Plus IDprimer set, 9947A genomic DNA, lx Fast Buffer I (Takara BIO USA Inc.,Madison, Wis.), 200 μM dNTPs and 0.315 U SpeedSTAR in a 10 μL reactionvolume. Cycling conditions for fast performance were set to: 1 min at95° C. (enzyme activation) followed by 28 cycles of 4 s at 98° C., 15 sat 59° C., 5 s at 72° C. and a 1 min at 72° C. final extension.

SpeedSTAR Biochip Reactions:

For biochip PCR the 7 μL reaction mix contained 1.4 μL Profiler Plus IDprimer set, 9947A genomic DNA, 1× Fast Buffer I buffer, 200 μM dNTPs and0.42 U SpeedSTAR. Cycling parameters were set to 70 s at 95° C., 28cycles of 4 s, 98° C.; 15 s, 59° C.; 7 s, 72° C. and a final extensionof 1:30 min at 70° C.

KOD Reactions:

Amplification with KOD were perfoiiiied with 2 μL Profiler Plus IDprimer set, lx KOD buffer (EMD Biosciences Inc., Gibbstown, N.J.), 200μM dNTPs, 1 ng 9947A genomic DNA, 1.5 mM MgSO₄, 0.2 U KOD in a 10 μLreaction volume. Cycling conditions were: 2 min, 95° C. followed by 28cycles of 4 s, 98° C.; 30 s, 59° C.; 10 s, 72° C. with a final extensionof 1 min, 72° C.

PyroStart Reactions:

Reaction mixtures with PyroStart in a lx final concentration alsocontained 2 μL Profiler Plus ID primer set and 1 ng 9947A genomic DNA ina 10 μL reaction and were cycled at: 1 min, 95° C. and 28 cycles of 4 s,98° C.; 20 s, 59° C.; 30 s, 72° C. followed by a final extension of 1min at 72° C.

Multiplex PCR with Other STR Typing Kits:

The suitability of SpeedSTAR to generate full. STR profiles with otherSTR typing kits (AmpFCSTR® Identifier® (Identifiler), AmpFlSTR® COfiler®PCR Amplification Kit (COfiler), (Applied Biosystems) was tested in tubeand biochip with the reaction conditions as described above forSpeedSTAR with the Profiler Plus ID kit. In these reactions, theProfiler Plus ID primer sets were replaced with the primer set from eachof the kits.

Reproducibility

Reproducibility studies in tube and biochip were performed with TaqGold™and SpeedSTAR using ing 9947A genomic DNA as a template. For tubereproducibility 5 individual reactions were prepared. Biochipreproducibility was determined in 3 biochip PCR runs with 8 reactionseach.

Sensitivity

Sensitivity studies for SpeedSTAR amplification in tube and biochip wereperformed using the following amounts of 9947A template DNA: In tube: 4ng, 2 ng, 1.5 ng, 1 ng, 0.5 ng, 0.25 ng, 0.125 ng, 0.1 ng, 0.05 ng, 0.03ng, 0.02 ng, 0.01 ng, 0.006 ng; in biochip: 4 ng, 2 ng, 1.5 ng, 1 ng,0.5 ng, 0.25 ng, 0.1 ng, 0.05 ng, 0.025 ng, 0.02 ng, 0.015 ng, 0.01 ng,0.006 ng. The reactions at each template level were performed induplicate.

STR Separation and Detection Instrumentation

Amplified products were separated and detected using the NetworkBiosystem Genebench-FX™ Series 100 (Pyzowski and Tan, Advances inBiochip-Based Analysis: A Rapid Field-Based Approach 59th Annual Meetingof the American Academy of Forensic Sciences San Antonio, Tex., Feb.19-24, 2007). This instrument was developed and optimized specificallyfor STR analysis. To 2.7 μL of each amplified product 10.2 μL formamideand 0.1 μL of Genescan 500 LIZ internal lane standard (both AppliedBiosystems, Foster City, Calif.) were added. After denaturation at 95°C. for 3 min and snap cooling on ice, samples were loaded into theseparation chip and electrophoretically moved into the separationchannels by applying a 350 V/cm electric field for 90 seconds. This wasfollowed by the application of a 150 V/cm electric field along theseparation channel to separate the DNA fragments. All separations werecarried out at 50° C.

Data Analysis

Data was analyzed with the GeneMarker® HID STR Human IdentificationSoftware, Version 1.51 (SoftGenetics LLC, State College, PA). Signalstrengths were noimalized to the internal lane standard and thepercentages of stutter, incomplete NTA as well as PHR were determined.PHR is calculated by dividing the lower signal strength allele by thehigher signal strength allele within the locus. The level of incompleteNTA is calculated by dividing the signal strength of the templatefragment (−A) by the signal strength of the adenylated fragment (+A).

Example 2 Temperature Profiles of Thermal Cycling Instruments andReaction Solutions in Conventional PCR Tubes and Microfluidic Biochips

Amplification reactions were performed in thin-walled PCR tubes using acommercial thermal cycler and in microfluidic biochips using thetheiival cycler of Example 1. For tube reactions, the EppendorfMastercyler™ was utilized. FIG. 2A shows the temperature of the blockand the reaction solution within a tube for one of the 28 thermal cyclesusing a conventional STR cycling protocol. The Mastercycler™ heating andcooling system is based on a heat pump with an integrated block for tubeinsertion. The time and temperature setpoints are 1 minute at 98° C. fordenaturation, 1 minute at 59° C. for annealing, and 1 minute at 72° C.for extension. A comparison of the temperature profiles for the heatblock and the reaction solution shows a lag in the response of thesolution temperature relative to the block temperature. The measuredheating and cooling rates of the block are 5.6° C./sec and 4.9° C./secand of the solution are 4.8° C./sec and 3.3° C./sec. The block makes thetemperature transition from extension (72° C.) to denaturation (98° C.)in 14 seconds, but the solution does not achieve the setpointtemperature for 39 seconds. Transitions between the denaturation andannealing steps (59° C.) take 10 and 27 seconds and between theannealing and extension steps take 7 and 24 seconds for the block andsolution respectively.

The temperature profiles of the Eppendorf Mastercyler™ block and thereaction solution for one of the 28 thermal cycles under fast cyclingconditions are shown in FIG. 2B. The time and temperature setpoints are5 seconds at 98° C. for denaturation, 15 seconds at 59° C. forannealing, and 5 seconds at 72° C. for extension. However, the delayedand dampened response of the solution prevents it from achieving thedesired setpoint temperatures.

The temperature profiles of the heat pump and the reaction solution forone of the 28 themial cycles for the thermal cycler of the inventionusing fast cycling conditions were also determined (FIG. 3). For thedetermination of the reaction solution temperature, a sensing chamberwithin the biochip was used. The time and temperature setpoints are 4seconds at 95° C. for denaturation, 15 seconds at 59° C. for annealing,and 7 seconds at 72° C. for extension. The measured heating and coolingrates of the heat pump are 21.5° C./sec and 21.7° C./sec, and themeasured heating and cooling rates of the reaction solution are 14.8°C./sec and 15.4° C./sec.

Accordingly, the thermal cycler of the invention is capable of heatingand cooling the reaction solution at a rate that is 3 to 5 times fasterthan the commercial block-based cycler. The transition times betweenextension, denaturation, and annealing steps for the heat pump are 1.7,2.1, and 0.7 seconds and for the solution 2.7, 4.5, and 2.2 seconds. Thethermal cycler of the invention allows the reaction solution to reachthe required temperatures approximately 7-fold faster than theblock-based cycler, resulting in defined and controlled incubationtemperatures and times under fast cycling conditions.

Example 3 Evaluation of PCR Enzymes in Tubes

A large number of polymerases were evaluated for potential use for fast,multiplexed. STR analysis, and candidates were selected based in part onhot-start activation time and extension rate. The reported properties ofthe four polymerases selected for experimental evaluation compared withrecommended conditions for TaqGold™ are presented in Table 1(A).

TABLE 1 A - Reported Polymerase characteristics Polymerase ATG SpeedSTARPyroStart KOD 3′-5′ Exonuclease activity No Yes, <20% No Yes Generationof 3′-dA Yes Yes, >80% Yes No overhangs Hot Start mechanism ChemicalAntibody Chemical Antibody modified modified Initial activation 95° C./95° C./ 95° C./ 95° C./ 11 min 1 min 1 min 2 min Elongation rate 16.67100-200 40 100 [nucleotides/sec] B - Optimized performance ofpolymerases in tube reactions Standard ATG Optimized ATG SpeedSTARCycling  95° C./11 min  95° C./11 min 95° C./1 min conditions 94° C./1min 98° C./10 sec 98° C./4 sec  59° C./1 min {close oversize bracket}×28 59° C./45 sec {close oversize bracket} ×28  59° C./15 sec {closeoversize bracket} ×28 72° C./1 min 72° C./30 sec 72° C./5 sec   60°C./45 min  72° C./15 min 72° C./1 min Amplification 145.1 min 71.67 min19.13 min time Signal Strength 561-1655 600-3876 945-3669 range [RFU]Stutter range 4.18-11.16%  3.91-13.77% 6.49-13.56% NTA range 1.54-7.67%1.93-20.13% 3.07-19.68% PHR 0.88-0.93%  0.74-0.96%  0.83-0.93% B -Optimized performance of polymerases in tube reactions PyroStart KODCycling 95° C./1 min 95° C./2 min conditions 98° C./4 sec  98° C./4 sec  59° C./20 sec {close oversize bracket} ×28  59° C./30 sec {closeoversize bracket} ×28  72° C./30 sec  72° C./10 sec 72° C./1 min 72°C./1 min Amplification 33.12 min 29.47 min time Signal Strength 751-31971091-3494 range [RFU] Stutter range 6.21-16.91% 4.46-23.61% NTA range3.01-23.09% PHR  0.71-0.94%  0.61-0.94%

The evaluated enzymes have reported extension rates ranging fromapproximately 15-200 nucleotides/second; in general, the reportedextension rates are based on singleplex amplifications and may besomewhat lower for multiplex applications.

PCR conditions in tubes were initially investigated for these fourenzymes with the goal of achieving full STR profiles in the least amountof time. For all reactions, 1 ng of human genomic DNA was amplifiedusing primer pairs from the Profiler Plus ID kit and vendor recommendedbuffers and enzyme concentrations, and resulting profiles wereseparated, detected, sized, and quantified using Genebench-FX™ Series100. Various times and temperatures for the denaturation, annealing, andextension steps were deteimined to give total times for PCRamplification with signal strengths suitable for STR interpretationranging from 19.13 minutes for SpeedSTAR to 71.7 minutes for TaqGold™[Table 1(B)]. Method conditions of the invention allow amplification tobe performed 2-10 fold more rapidly than recommended conditions forTaqGold™.

Further evaluation of the enzymes for forensically relevant performanceincludes signal strength, levels of stutter and incomplete NTA and PHR[Table 1(B)]. All enzymes are capable of performing highly multiplexedamplification using the Profiler Plus ID primers. Signal strengths forSpeedSTAR, Pyrostart, KOD, and optimized TaqGold™ reactions are alleither approximately the same or higher than those generated usingstandard TaqGold™ PCR conditions.

With respect to incomplete NTA, both SpeedSTAR and PyroStart as well asoptimized TaqGold™ reactions exhibited levels that are up to three timeshigher than that of standard TaqGold™ reactions. For most alleles,levels fall below the 15% interpretation threshold. Those alleles havinghigher levels of incomplete NTA can be decreased to below the 15%interpretation threshold as discussed below. The KOD polymerasepossesses 3′-5′ exonuclease activity and does not generate fragmentswith A-overhangs; accordingly, all alleles were 1 nucleotide shorterthan their allelic ladder counterpart.

The relative levels of stutter observed for optimized TaqGold™,SpeedSTAR and PyroStart reactions are similar to the range of stutterproduced with standard TaqGold™ reactions; the range of stuttergenerated with KOD is slightly higher than the stutter for standardTaqGold™.

Example 4a Fast PCR Protocol Using SpeedSTAR Polymerase in Tubes andBiochips

Based on the results presented above, the SpeedSTAR polymerase wasselected for further evaluation in biochips with the goal of minimizingthe total cycling time and achieving full STR profiles that satisfysignal strength, PHR, incomplete NTA and stutter interpretationrequirements.

The time and temperature setpoints for amplification using SpeedSTAR inthe microfluidic 16-sample biochip on the thermal cycler of theinvention are 70 seconds 95° C. for hot-start activation followed by 28cycles of 4 seconds at 95° C. for denaturation, 15 seconds at 59° C. forannealing, and 7 seconds at 72° C. for extension. A final extension of90 seconds at 72° C. is performed for a total protocol time of 17.3minutes. Tube reactions in the Eppendorf Mastercycler were performed in19.13 minutes comprising of block times and temperatures set to aninitial activation time of 1 minute at 95° C., 28 cycles of 4 seconds at98° C., 15 seconds at 59° C. and 5 seconds at 72° C. followed by a finalextension of 1 minute at 72° C.

FIGS. 4A and 4B show STR profiles generated with the preceding SpeedSTARcycling conditions in (FIG. 4A) 7 μL biochip and (FIG. 4B) 10 μL tubereactions using 0.5 ng of DNA and Table 2 presents signal strengths forall Profiler Plus ID alleles from the SpeedSTAR biochip and tubereactions as well as TaqGold^(rm) in tubes using the standard protocol.Signal strengths of the 0.5 ng SpeedSTAR biochip reactions are onaverage approximately 2 times higher than those of the Ing standardTaqGold™ reactions, while the signal strengths of the 0.5 ng SpeedSTARtube reactions are on average approximately the same as those of theTaqGold™ reactions.

TABLE 2 Comparison of signal strength, PHR, NTA and stutter forSpeedSTAR biochip and tube reactions and ATG standard reactionsSpeedSTAR biochip reaction SpeedSTAR tube reaction Signal Stutter SignalStutter Locus Allele strength PHR NTA (%) (%) strength PHR NTA (%) (%)D3S1358 14 1404.218 0.79 9.08 727.11 0.81 9.74 D3S1358 15 1110.915 8.0510.35 895.12 10.50 8.14 vWA 17 2338.633 0.95 6.14 1346.81 0.65 6.62 vWA18 2216.64 6.83 12.87 875.84 6.78 11.96 FGA 23 747.5319 0.87 4.21 877.220.96 2.54 FGA 24 648.8992 3.34 9.72 840.03 2.30 8.32 Amelogenin X5645.423 4.04 1324.78 2.02 D8S1179 13 3784.38 5.62 10.70 1012.17 5.4711.56 D21S11 30 2008.992 3.15 13.05 787.70 3.45 13.29 D18S51 15 1655.9910.70 6.90 11.44 924.04 0.72 5.48 9.09 D18S51 19 1157.636 9.53 14.13663.76 8.92 13.90 D5S818 11 2904.473 7.56 9.65 1608.46 3.97 8.73 D13S31711 3906.373 3.32 6.78 1867.36 3.21 7.74 D7S820 10 1232.908 0.93 7.23647.24 0.73 8.01 D7S820 11 1149.849 10.16 7.37 469.59 10.00 5.96Standard ATG reactions Signal strength PHR NTA (%) Stutter Locus AlleleAverage STDEV Average STDEV Average STDEV Average STDEV D3S1358 14831.69 167.25 0.92 0.04 1.88 0.52 D3S1358 15 802.99 141.78 1.54 0.345.31 0.23 vWA 17 765.61 83.69 0.90 0.08 4.22 0.44 vWA 18 736.85 88.404.23 0.19 7.38 0.48 FGA 23 735.41 130.32 0.93 0.05 2.14 0.45 FGA 24727.02 122.82 1.98 0.44 7.19 0.43 Amelogenin X 1655.65 343.77 3.35 0.49D8S1179 13 1405.57 105.18 4.52 0.58 6.51 0.62 D21S11 30 1280.31 81.303.00 0.66 7.33 0.60 D18S51 15 879.42 137.76 0.88 0.05 4.68 0.74 7.890.80 D18S51 19 814.14 125.75 5.95 0.68 11.16 0.52 D5S818 11 1599.08223.78 3.71 0.17 4.94 0.44 D13S317 11 1486.50 196.47 2.33 0.28 4.18 0.47D7S820 10 634.96 113.82 0.89 0.08 4.69 0.99 D7S820 11 561.37 92.47 7.670.72 4.57 0.62

Example 4b Allele Characterization of Fast PCR Using SpeedSTARPolymerase in Tubes and Biochips

In order to characterize the products of fast PCR reactions from Example4a, quantification of PHR, incomplete NTA and stutter was performed.Biochip and tube reactions using the SpeedSTAR polymerase show moreinter-locus peak height imbalance compared to that for the TagGold™reactions. The PHR for alleles generated in biochip reactions is between0.70 and 0.95 and is approximately the same in tubes; all fall withinacceptable interpretation guidelines. Reactions using SpeedSTAR have PHRthat are approximately 10% lower than those determined for standardTaqGold™ reactions. Similarly, the level of incomplete NTA for mostalleles in both biochip and tube reactions using SpeedSTAR areapproximately the same (2.0 and 10.6%); both are approximately 2 timeshigher than for TaqGold™ control reactions. The exception is incompleteNTA for the D3S1358 alleles, which is 4.8 to 7 times higher withSpeedSTAR than with TaqGold™; even in this case, the level of incompleteNTA is below 12% for the SpeedSTAR enzyme. Finally, the level of stutterin both biochip and tube based reactions using SpeedSTAR is betweenapproximately 6.0 and 14.1%, on average approximately 1.6-fold higherthan that for standard TagGold™ tube reactions.

The microfluidic biochip reactions using 0.5 ng template DNA generatesignal strengths that are approximately 2 times higher than those forstandard TaqGold™ reactions using 1 ng template. This result suggeststhat the SpeedSTAR enzyme in the biochip and the TaqGold™ enzyme in theconventional reaction act with similar efficiencies; the DNAconcentration in the biochip is approximately 1.8-fold that in the tube,which corresponds to the 2 fold greater in signal strength. In contrast,the fast tube based reactions are less efficient than the controlTaqGold™ reactions; the approximately 40% reduction in product yield islikely the consequence of the poor cycling profile that results whencommercial thermal cyclers are used for fast thermal cycling. Even inthis circumstance, signal strengths are well over the levels requiredfor interpretation and can be raised significantly by increasing theextension time by a few seconds per cycle (data not shown).Repeatability and reproducibility of the signal strength for fast PCRreactions in biochips and tubes are similar to those in conventionalreactions.

The inter-locus allele signal strength for the fast biochip and tubereactions shows a higher level of imbalance as compared to the TaqGold™reaction. The inter-loci signal strength balance is influenced bynumerous factors including primer concentration, annealing temperatureand time, and molecular weight of the loci. The STR amplification kitused for these experiments has a set of primer concentrations that areoptimized for the TaqGold™ enzyme and the recommended cycling protocols.The signal strengths of the loci can be modified by adjusting the primerconcentrations utilized in the amplification reactions (Henegariu etal., Biotechniques 1997, 23, 504-11).

The relationship between signal strength and template level for fastbiochip and tube reactions is as expected with signal strength generallyincreasing with template. Good peak morphology is observed for allalleles at high template levels of 4 ng (which generate alleles withsimal strengths of greater than 12000 RFU). At template levels of 0.03ng and below some allele drop-out occurs. This effect is observed whenamplification reactions are carried out with limited number of templateDNA strands in the solution leading to stochastic amplification (Walshet al., PCR Methods Appl. 1992, 1, 241-250). The presence of readilydetectable signal for both the high-signal strength alleles andlow-signal strength alleles at a template level of 0.006 ng,demonstrates the high sensitivity of the fast biochip and tube reactioncoupled with Genebench-FX™ Series 100 separation and detection,demonstrating the utility of this system for low copy number analysis.Taken together, this data also suggests that the fast PCR approach andthe thermal cycler of the invention and Genebench instrumentation have ahigh template dynamic range.

Example 4c DNA Template Levels and Allele Characteristics in Fast PCRReactions

The effects of template DNA on signal strength for fast PCR reactionsusing SpeedSTAR polymerase in (FIG. 5A) biochip and (FIG. 5B) tubereactions are presented in FIGS. 5A and 5B. The alleles selected foranalysis were Amelogenin, the allele with the highest signal level inthe STR profile and FGA 23 and 24 and D7S820 10 and 11, the alleles withthe lowest signal levels in the profile. Signal strengths for allalleles increase as the DNA template level increases from 0.006 ng to 4ng in both SpeedSTAR biochip and tube reactions. At a template level of0.006 ng, signal strengths for the amelogenin peak of 111 RFU forbiochip and 58 RFU for tube reactions were observed. At a template levelof 4 ng, signal strengths of 12680 RFU were seen for biochip and 5570RFU for tube reactions. All alleles observed in both reaction typesshowed good peak morphology.

For fast biochip reactions (FIG. 6A), PHR is between 0.6 and 1.0 fortemplate levels ranging from 0.05 to 4.0 ng. For template levels below0.05 ng, PHR decreases until 0.025 ng, when instances of allelicdropouts occur and PHR of zero are observed. Similar results areobserved for fast tube reactions, although they generally exhibitsomewhat lower PHR than biochips reactions (FIG. 6B). For biochipreactions, the level of incomplete NTA is 15% or less for templatelevels of 2.0 ng and below. For tube reactions, the incomplete NTAlevels surpass 15% at template levels of 1 ng and increase dramaticallyby 4 ng.

Two major differences between biochip and tube reactions are thetemperature profiles of the reaction solutions and the relativeconcentration of template and polymerase. The level of incomplete NTAdecreases as more polymerase is available. For biochip reactionsexperimental data show that over a DNA template range from 0.5-4.0 ngthe level of incomplete NTA decreases by approximately 50% as the amountof SpeedSTAR polymerase increases from 0.3U to 1.2U (FIGS. 7A and 7B).The level of stutter for fast biochip and tube reactions was relativelyconstant and generally less than 15% for all alleles over a templatelevel range of 0.25-4.0 ng (FIGS. 8A and 8B).

The speed of an STR amplification reaction is only relevant if thereaction itself generates actionable data that meets forensicinterpretation guidelines. The FBI has general guidelines that are usedfor STR interpretation, and individual laboratories set thresholds thatmust be met before a profile can be considered acceptable based on theirvalidation work (Holt et al., J. Forensic Sci. 2002, 47(1), p. 66-96;LaFountain et al., J. Forensic Sci., 2001, 46(5), 1191-8).

The conditions presented herein can generate fast STR profiles that meetthese guidelines. PHR for 0.5 ng template in biochip and tube reactionsmeet with the interpretation guidelines that state a level of 0.6 orgreater is required and are consistent with previously reported results(Leclair et al., J. Forensic Sci. 2004, 49, 968-80). For higher DNAtemplate amounts, PHR remain relatively constant but are lower thanthose for the ing TaqGold™ reactions. For low copy numbers, the PHR isdominated by amplification due to stochastic fluctuations.

The level of incomplete NTA is based on the ability of the polymerase tofully adenylate all STR amplicons. For conventional amplification, thisis accomplished by attaching a “pigtail” to the primer and increasingthe final extension time. The level of incomplete NTA for 0.5 ngbiochips and tube reactions described herein are within interpretationguidelines.

Levels of incomplete NTA increase with increasing DNA template (aconsequence of the increasing ratio of DNA to polymerase) and can bereduced by increasing the amount of polymerase, extension time percycle, and final extension time. The later two approaches are notwell-suited to fast multiplexed amplification, as they increase thereaction time. Increasing polymerase concentration is effective andcompatible with fast PCR. Stutter is a result of DNA strand slippageduring extension (Walsh et al., Nucleic Acids Res. 1996, 24(14),2807-12). The level of stutter described here for the 0.5 ng biochip andtube reactions fall within the interpretational guidelines and are alsoconsistent with previously cited reports. As expected, the level ofstutter appears to be independent of the DNA template level.

Example 4d Repeatability and Reproducibility Studies

The repeatability and reproducibility of the fast biochip (Table 3A) andtube (Table 3B) reactions using the SpeedSTAR polymerase were evaluatedby performing 24 identical PCR reactions in 3 PCR biochips and byperforming 5 identical tube reactions. For biochip reactions, theconfidence value (CV) for signal strength ranges from 17 to 24% and fortube reactions from 15 to 34%. The CV for standard TaqGold™ reactions isbetween 6 and 21%.

TABLE 3 Reproducibility of SpeedSTAR biochip (3A) and tube (3B)reactions PHR Signal Strength (RFU) Aver- NTA (%) Stutter (%) AlleleAverage STDEV Max Min age STDEV Max Min Average STDEV Max Min AverageSTDEV Max Min A: SpeedSTAR biochip reactions D3S1358.14 1235.40 271.431963.95 781.79 0.85 0.11 1.00 0.63 9.11 1.77 11.86 6.64 D3S1358.151066.55 251.75 1793.70 689.23 8.97 1.80 12.08 6.53 8.78 0.63 9.84 7.67vWA.17 2765.49 551.47 3818.46 1732.31 0.81 0.12 0.96 0.56 6.19 1.27 8.254.76 vWA.18 2274.33 398.37 3197.88 1379.49 6.22 1.34 8.29 4.71 12.950.66 14.35 11.78 FGA.23 884.74 153.51 1276.87 620.51 0.84 0.11 0.99 0.612.51 0.58 3.51 1.03 FGA.24 794.52 176.07 1109 30 376.41 2.68 0.48 3.521.71 10.10 0.95 12.18 8.63 AMEL 5085.29 1138.51 7276.15 3271.82 3.700.40 4.54 3.01 D8S1179.13 3787.27 778.68 5700.08 2420.29 7.35 0.74 8.826.17 10.96 0.53 12.08 10.01 D21S11.30 1914.04 424.28 2725.91 1005.452.91 0.61 4.14 2.04 12.74 0.85 14.44 10.73 D18S51.15 2248.76 429.943056.15 1475.43 5.80 2.08 7.99 0.00 10.61 0.64 11.54 9.04 D18S51.191843.92 358.13 2549.69 1271.08 0.83 0.10 0.99 0.59 5.90 2.23 8.19 0.0014.52 0.85 16.64 12.96 D5S818.11 2734.70 500.56 3646.65 1629.74 9.041.88 12.07 6.34 8.90 0.33 9.89 8.35 D13S317.11 4097.95 701.10 5279.962724.10 3.02 0.85 6.39 2.25 6.87 0.42 7.95 6.09 D7S820.10 1877.24 384.593262.80 1397.95 0.84 0.09 1.00 0.69 6.68 0.60 7.67 5.67 D7S820.111594.27 309.99 2336.39 1124.30 10.60 0.68 11.81 9.19 7.26 0.47 7.99 6.42CV Min: 17% CV Min: 11% CV Min: 6% CV Min: 4% CV Max: 24% CV Max: 14% CVMax: 28% CV Max: 9% B. SpeedSTAR tube reactions D3S1358.14 1718.86464.62 2321.62 1249.71 0.86 0.08 0.94 0.74 19.51 0.44 20.04 18.84D3S1358.15 1455.28 285.03 1719.04 1110.96 19.68 0.78 20.71 18.56 8.340.49 8.88 7.65 vWA.17 1934.44 299.00 2150.18 1483.24 0.83 0.11 0.98 0.7112.12 0.22 12.32 11.75 vWA.18 1722.47 450.99 2439.38 1257.89 12.04 0.2712.29 11.65 12.72 0.50 13.37 12.18 FGA.23 1625.37 289.29 2020.33 1275.750.91 0.08 0.99 0.80 3.57 0.21 3.84 3.28 FGA.24 1561.32 346.77 2003.501185.22 3.07 0.21 3.30 2.75 9.57 0.53 10.22 8.90 AMEL 3669.80 844.564967.47 2799.39 5.02 0.27 5.41 4.68 D8S1179.13 2447.48 658.60 3268.311820.49 8.20 0.40 8.61 7.58 10.47 0.45 10.86 9.72 D21S11.30 1628.98436.18 2147.27 1222.17 4.85 0.33 5.26 4.45 11.78 0.28 12.14 11.54D18S51.15 1603.23 545.46 2261.97 1066.52 0.79 0.17 0.95 0.61 8.30 0.348.67 7.91 9.67 0.31 10.09 9.30 D18S51.19 1296.20 434.98 2043.27 963.188.79 0.99 10.49 7.91 13.56 0.88 14.69 12.28 D5S818.11 3412.19 894.754629.47 2506.84 6.27 0.17 6.47 6.02 8.38 0.31 8.86 8.03 D13S317.113093.19 749.20 3987.13 2204.34 3.94 0.14 4.16 3.81 6.49 0.25 6.86 6.25D7S820.10 1009.46 228.75 1297.92 780.76 0.93 0.03 0.98 0.90 7.86 0.658.55 6.86 D7S820.11 945.50 208.75 1228.39 705.49 12.13 1.53 14.38 10.566.52 0.34 7.02 6.15 CV Min: 15% CV Min: 3% CV Min: 2% CV Min: 2% CV Max:34% CV Max: 21% CV Max: 13% CV Max: 6%

The CV for PHR is up to 14% for biochips and 21% for tube reactions,compared to the 5% to 10% range observed for standard TaqGold™reactions. CVs for incomplete NTA in biochip reactions vary between 6%and 28%, and for tube reactions between 2 to 13%. Again, thesevariations are similar to the 4 to 28% range observed for standardTaqGold™ reactions. The CVs for stutter in biochips are 4 to 9%, intubes 2 to 6%, and are also similar to the range of 4-13% observed forstandard TaqGold™ reactions.

Example 4e

Compatibility with Other Commercially Available STR Kits

Using the same fast biochip and tube conditions described above, aseries of samples were evaluated using primer sets from the COfiler™ andIdentifiler™ kits. FIGS. 9A and 9B show the achievement of full profilesusing these primer sets using the thermal cycler of the invention, theSpeedSTAR enzyme, and the protocols described herein. Each is suitablefor these commercially available kits as well. Although full profileswere achieved, imbalance in the signal strengths across the loci wasobserved.

Example 5

Fast Sequencing with the Thermal Cycler

The thermal cycling instrument and methodology can also be applied tofast DNA sequencing reactions. In this implementation of the fastthermal cycler, the instrumentation and biochip are the same as thatused for PCR. Different reaction solutions, polymerase and cyclingtemperatures are applied for the sequencing reaction. The currentlycommercially available sequencing reactions take 49 min (for GE AmershamDYEnamic™ ET Terminato Cycle Sequencing Kit) and 2.25 hr (for AB Big DyeV3.1. Utilizing the NetBio thermal cycler, with conventional reagents,allowed the sequencing reaction time to be reduced to 21 minutes.

Fast sequencing has been achieved using the thermocycler disclosedherein and biochip comprising 16 lanes. The final reaction volume in thechips was 7 μL. Half strength sequencing reactions were set up with theDYEnamic™ ET Terminator Cycle Sequencing Kit from GE Healthcarefollowing the manufacturer's protocol. All volumes were scaled downaccordingly to accommodate the 7 μL final volume. Template for thereaction was 0.1 μmol B. subtilus with a fragment size of 343 bp.

Three cycling protocols were demonstrated with the first protocolconsisting of 30 cycles of (20 s at 95° C., 15 s at 50° C. and 60 s at60° C.) (total cycling time is 51.7 min), the second protocol consistingof 30 cycles of 5 s at 95° C., 15 s at 50° C. and 30 sat 60° C. (totalcycling time is 29 min) and a third protocol consisting of 30 cycles of5 sat 95° C., 10 sat 50° C. and 20 s at 60° C. (total cycling time of21.6 min). Each sequencing reaction was cleaned-up with ethanolprecipitated, and separated on Genebench FX Series 100. The averagePHRED scores for sequencing a 343 bp PCR product for the 3 cyclingprotocols were 282, 287, and 279 respectively; demonstrating thatsequencing of a 343 bp product can be achieved in chip in less than 22min. FIG. 10 shows the DNA sequence of the fast DNA sequencing protocol.

In general, biochip based multiplexed amplification of one or morenucleic acids utilizing the systems and methods described herein havethe advantage of providing amplified nucleic acid products insignificantly shorter overall reaction times with respect to reactionsrun in thin-walled tubes and utilizing presently commercialized thermalcycling units.

1-69 (canceled)
 70. A method for simultaneously amplifying of aplurality of loci in a nucleic acid solution comprising: providing in asingle solutions contained in at least two reaction chambers located ina biochip, samples having at least ten target nucleic acid loci to beamplified, with at least ten different primer pairs, each primer pairhybridizing to one of the at least ten loci to be amplified, saidsolution further comprising: (i) one or more buffers; (ii) one or moresalts; (iii) a nucleic acid polymerase; and (iv) nucleotides; andproviding a thermal control system comprised of (i) a TCE having a firstsurface in thermal communication with said at least two reactionchambers, said TCE further comprising a means for heating and cooling,(ii) at least one thermosensor, (iii) a controller that receives signalsfrom said at least one thermosensor and (iv)a power supply, said atleast one thermosensor positioned and configured to measure theeffective temperature of each of the single solutions in the reactionchambers of the biochip and to provide feedback to the TCE to heat orcool the solution to set or maintain the solution at a desiredtemperature; obtaining, with said controller of the thermal controlsystem, a target sample solution temperature, from a control algorithmthat stores the target sample temperature, for a sample for a firstsub-step of a plurality of predetermined processing sub-steps, to beperformed on the samples, where in the first sub-step of the processingstep is one of a denaturing, an annealing or an extending of areplication of a polymerase chain reaction; obtaining with saidcontroller from said control algorithm, a time and temperature profilerepresenting a comparison between the target sample temperature asmeasured by said thermosensor in thermal communication with said atleast two reaction chambers, and a plurality of different temperaturesfor said TCE, wherein the plurality of different temperatures aredifferent from each other and different from the target sampletemperature and correspond to different time points, of a predeterminedset of time points, of the first sub-step; identifying, with thecontroller from said control algorithm, for a first time point of thedifferent time points for the first sub-step, a first temperature of theplurality of different temperatures using the time and temperatureprofile; and controlling, with the controller from said controlalgorithm, a temperature of the TCE at the first time point of the firstsub-step to be the first temperature, wherein the TCE is in thermalcommunication with said at least two reaction chambers and controls atemperature of said samples.
 71. The method of claim 70, furthercomprising: identifying, for a second time point of the different timepoints for the first sub-step, a second temperature of the plurality ofdifferent temperatures using the time and temperature profile; andcontrolling the temperature of the TCE at the second time point of thesub-step to be the second temperature.
 72. The method of claim 71,further comprising: maintaining a temperature of at least one reactionchamber at approximately a same temperature for the first and secondtime points of the sub-step by changing the temperature of the TCE fromthe first temperature at the first time point to the second temperatureat the second time point.
 73. The method of claim 71 further comprising:identifying, for at least one additional time point of the differenttime points, at least one additional temperature of the plurality ofdifferent temperatures using the time and temperature profile and theplurality of different temperatures and the at least one additional timepoint; and controlling the temperature of the TCE at the at least oneadditional time point of the sub-step to be the at least one additionaltemperature.
 74. The method of claim 73 wherein the controlling of thetemperature includes gradually changing the temperature from the secondtemperature to the at least one additional temperature.
 75. The methodof claim 73 further comprising: maintaining a temperature of the atleast one reaction chamber at approximately a same temperature for thefirst, the second and the at least one additional time points of thesub-step by changing the temperature of the TCE from the firsttemperature at the first time point to the second temperature at thesecond time point to the at least one additional temperature at the atleast one additional time point.
 76. The method of claim 75 whereinmaintaining the temperature of the at least one reaction chambermaintains an approximately constant temperature of the sample.
 77. Themethod of claim 76 wherein the approximately constant temperature is atemperature from a group consisting of 95 degrees; 59 degrees; and 72degrees Celsius.
 78. The method of claim 70 wherein the processing stepis a DNA sequencing step.
 79. The method of claim 70, wherein for eachof the denaturing, the annealing and the extending of replication, thetemperature of the sample is approximately constant.
 80. The method ofclaim 70, further comprising: controlling, with the controller, thetemperature of the TCE to set a temperature of a reaction chamber at thetarget sample temperature; measuring with the at least one thermosensorpositioned and configured to measure the effective temperatures of eachof the single solutions in the reaction chambers, a temperature of theat least two reaction chambers concurrently with controlling thetemperature of the TCE which results in the temperature of each of thesingle solutions in the reaction chambers being at the target sampletemperature.
 81. The method of claim 70 further comprising: obtaining atleast one additional target sample temperature wherein the time andtemperature profile provides at least one additional target sampletemperature and at least one additional set of temperatures of the TCEfor at least one additional sub-step of the processing step;identifying, for a time point of the at least one additional sub-step, atemperature of the at least one additional set of temperatures using atleast one additional time and temperature profile generated using the atleast one additional target sample temperatures and the at least one setof additional temperatures and the time point of the at least oneadditional sub-set; and controlling the temperature of the TCE at thetime point of the at least one additional sub-set at the at least oneadditional set of temperatures.
 82. An apparatus comprising: acontroller of a thermal control system configured to: obtain a targetsample temperature, from a controller that stores the target sampletemperature, for a sample for a first sub-step, of a plurality ofpredetermined sub-steps, of a processing step, of a plurality ofpredetermined processing steps, to be performed on the sample, whereinthe first sub-step of the processing step is one of a denaturing, anannealing or an extending of a replication of a polymerase chainreaction sub-step; obtain a time and temperature profile, from thecontroller wherein the profile provides a correlation between the targetsample temperature, as measured by a thermosensor in thermalcommunication with a reaction chamber, with a target sample temperatureapplied to the reaction chamber, and a plurality of differenttemperatures for a TCE, wherein the plurality of different temperaturesare different from each other and different from the target sampletemperature and correspond to different time points, of a predeterminedset of time points, of the firsts sub-step; identify, for a first timepoint of the different time points for the first sub-step, a firsttemperature of the plurality of different temperatures using atemperature profile between the target sample temperature and theplurality of different temperatures and the first time point; andcontrol a temperature of the TCE at the first time point of the firstsub-step to be the first temperature; wherein the TCE is in thermalcommunication with a reaction chamber and controls a temperature of thesample.